Biomarker for detecting cancer

ABSTRACT

Methods for detecting colorectal cancer or precancerous conditions such as adenomatous polyps in a subject by detection of a galactose-containing 40-kDa molecule in a serum sample from the subject are provided. Methods for quantifying the amount of a galactose-containing molecule in a serum sample are also provided. The methods can further comprise assaying the sample for the quantity of at least one additional marker to confirm the detection or diagnosis of colorectal cancer using one or more additional quantitative immune-detection assays; the additional marker can be at least one of galectin-3, a peptide derived from galectin-3, carcinoembryonic antigen (CEA), and CYFRA21-1.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/656,390, filed Apr. 12, 2018, which is incorporatedherein by reference in its entirety.

This invention was made with government support under grant numbersCA069480 and CA068400 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of molecularbiology, immunology and oncology. More particularly, it concerns methodsfor detecting biomarkers linked to the development of cancer, andadenomatous polyps that may develop into cancer.

2. Description of Related Art

Modern medicine has developed an arsenal of therapies that can bebrought to bear against cancer. However, a major factor in theeffectiveness of such therapies is the stage of the cancer beingtreated. Late stage cancers and cancer cells that have alreadymetastasized from their site of origin remain difficult to treat andcontinue to result in high rates of mortality among patients. Hencesuccessful anti-cancer therapy is largely dependent upon early andaccurate diagnosis. Many cancer diagnostic techniques, however, areinaccurate or invasive, reducing the opportunity for early detection andsuccessful treatment. For example, in the case of colorectal cancer theprimary diagnostic technique, colonoscopy, is viewed as highly invasiveand typically only applied every 5-10 years and only after the age of50. Thus, there remains a need for accurate and non-invasive techniquesfor cancer detection.

In the United States, colonoscopy is the dominant strategy for coloncancer screening. However, in all other countries (including Canada, theUnited Kingdom, Australia and most of Europe) where programmatic coloncancer screening takes place, the screening process is typically atwo-step process. Usually a stool test (fecal immunochemical testing orFIT) is performed and if positive, then a colonoscopy takes place. Ablood test which replaces FIT in the two-step screening process,therefore, would be extremely useful in increasing the sensitivity andselectivity of the screening process.

Additionally, there is a considerable advantage in detectingprecancerous lesions, including, but not limited to, advanced adenomas.An effective method of detecting such precancerous lesions would lead tothe ability to remove such lesions before they become cancerous, whichwould be easier and less risky for the patients.

Accordingly, there is an unmet need for an improved blood test for thedetection of colorectal cancer and precancerous lesions in theintestinal tract.

SUMMARY OF THE INVENTION

Methods according to the present invention meet this need for animproved blood test for the detection of precancerous lesions and cancerin the colorectal tract with a test that provides high sensitivity andspecificity.

In a first embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subject;(b) desialylating the serum; (c) contacting the desialylated serum withan antibody that binds haptoglobin or a cancer-associated haptoglobinglycoform to form a complex, wherein the complex comprises the antibodyand a galactose-containing molecule; and (d) detecting thegalactose-containing molecule on the complex with a detectable lectinthat binds galactose, wherein an elevated level of thegalactose-containing molecule detected as compared with a control levelis indicative of a colorectal cancer. In certain aspects, a method ofthe embodiments is defined as an in vitro method. For example, detectionmethod of the embodiments may employ assays such as those disclosed inPCT Application Serial No. PCT/US2012/059567 by Bresalier et al.,entitled “Biomarker for Detecting Cancer and U.S. patent applicationSer. No. 15/726,123, which are incorporated herein by reference in theirentirety.

In a further embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subject;(b) desialylating the serum; (c) contacting the desialylated serum withan antibody that binds haptoglobin to form a complex, wherein thecomplex comprises the antibody and a galactose-containing molecule; (d)detecting the galactose-containing molecule on the complex with adetectable lectin that binds galactose; and (e) determining the presenceof (or the probability of the presence of) a colorectal cancer based onan elevated level of the galactose-containing molecule detected ascompared with a control level. In certain aspects, a method of theembodiments further comprises performing or obtaining the results of oneor more secondary tests to determine the presence of a colorectalcancer. For example, the secondary test can be, without limitation, afecal occult blood test (FOBT), a colonoscopy or a secondary blood test(e.g., a test for levels of carcinoembryonic antigen (CEA) in theblood). In some alternatives, as described below, additional biomarkerscan be used in addition to CEA or in place of CEA.

In still a further embodiment there is provided a method for quantifyinga galactose-containing molecule in a serum sample comprising (a)obtaining a serum sample of a subject; (b) desialylating serum; (c)contacting the desialylated serum with an antibody that bindshaptoglobin to form a complex, wherein the complex comprises theantibody and a galactose-containing molecule; and (d) detectinggalactose on the complex with a detectable lectin that binds galactose,thereby quantifying the galactose-containing molecule in the sample.

In certain aspects of the embodiments, step (b) further comprisesdiluting the serum between about 25-fold and 150,000-fold, 100-fold and100,000-fold, 1,000-fold and 50,000-fold, or 10,000-fold and 50,000-fold(e.g., at least about 20-fold, 25-fold, 100-fold, 500-fold, 1,000-fold,5,000-fold, 10,000-fold or 15,000-fold) before desialylating the dilutedserum. As used herein, “diluting” means mixing of a sample with a largervolume of a second non-sample solution (e.g., water). As used herein“fold dilution” refers to the dilution factor as compared to a serumsample that has not been mixed with any additional solution (i.e., anunadulterated serum sample).

In a further embodiment there is provided a method for detecting acolorectal cancer, comprising (a) obtaining a serum sample of a subjectwherein the serum sample has been diluted between 25-fold and150,000-fold; (b) desialylating the diluted serum; (c) contacting thedesialylated serum with an antibody that binds haptoglobin to form acomplex, wherein the complex comprises the antibody and agalactose-containing molecule; and (d) detecting galactose-containingmolecule on the complex with a detectable lectin that binds galactose,wherein the elevated level of the galactose-containing molecule detectedas compared with a control level is indicative of a colorectal cancer.

In still a further embodiment there is provided a method for quantifyinga galactose-containing molecule in a serum sample comprising (a)obtaining a serum sample of a subject wherein the serum sample has beendiluted between 25-fold and 150,000-fold; (b) desialylating the dilutedserum; (c) contacting the desialylated serum with an antibody that bindshaptoglobin to form a complex, wherein the complex comprises theantibody and a galactose-containing molecule; and (d) detectinggalactose-containing molecule on the complex with a detectable lectinthat binds galactose, thereby quantifying the galactose-containingmolecule in the sample.

Thus, in certain aspects a method is provided for quantifying agalactose-containing molecule in a serum sample in accordance with theforegoing embodiments wherein an elevated level of thegalactose-containing molecule detected as compared with a control levelindicates that the subject has either: (i) an elevated risk ofcolorectal cancer, such as is associated with a precancerous condition;or (ii) actually has colorectal cancer. Thus, in some aspects, a methodof detecting colorectal cancer is provided comprising administering atleast a second test for colorectal cancer to a subject identified ashaving a risk for colorectal cancer. For example, the second test can bea blood test, a stool test, or a colonoscopy.

Thus, in some aspects, obtaining a serum sample comprises obtaining adiluted serum sample. For example, a serum sample can be a sample thathas been diluted by at least about 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-foldor 15,000-fold.

Certain aspects of the embodiments concern obtaining a serum sample froma patient. The serum sample can, for example, be directly obtained bydrawing blood from a patient. In certain cases the sample is obtainedfrom a third party (e.g., a doctor or clinic) or is a frozen bankedsample.

In certain aspects methods of the embodiments concern desialylating adiluted serum sample. For example, the sample subjected to desialylationcan be a sample that has been diluted by about or at least about25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold or 15,000-fold prior todesialylation. A diluted sample can be desialylated by a variety ofmethods, such as by treatment with an enzyme or a mild acid. Forexample, a diluted serum sample can be treated with a neuraminidaseenzyme (e.g., a Vibrio cholerae neuraminidase). In certain aspects, adiluted sample is treated with a mild acid such as H²SO₄, and optionallyheating the sample. For example, a diluted serum sample can be treatedwith 0.5 N H₂SO₄, and heated to about or at most about 50° C., 60° C.,70° C., or 80° C. for a period of about or at least about 30 min, 60min, or 90 min.

Certain aspects of the embodiments involve contacting a desialylated anddiluted serum with an antibody. For example, in some aspects the sampleis diluted to between 25-fold and 150,000-fold, 100-fold and100,000-fold, 1,000-fold and 100,000-fold, 10,000-fold and 100,000-foldor 15,000-fold and 150,000-fold (e.g., about a 20,000-fold dilution)before contacting the serum with an antibody. Thus, in certain aspects asample is diluted before desialylation, after desialylation or both.

Antibodies for use according to the embodiments include, withoutlimitation, polyclonal and monoclonal antibodies that bind tohaptoglobin (e.g., human haptoglobin). For example, the antibody can bean antibody that was raised against a purified human haptoglobin, suchas a rabbit polyclonal antiserum (e.g., the H-8636 antisera availablefrom Sigma). An anti-haptoglobin antibody, can in some cases, be labeledor immobilized. For example, in some aspects, the antibody isimmobilized on a bead (e.g., a magnetic bead) or a surface (e.g., in awell of a plate).

Some aspects of the embodiments involve a step of washing a complexcomprising the antibody and a galactose-containing molecule with a washsolution (i.e., contacting the complex with a volume of wash solution).For example, washing a complex comprising the antibody and agalactose-containing molecule can comprise washing the complex 1, 2, 3,4 or more times with a solution comprising a physiological amount ofsalt and a physiological pH, such as PBS. Optionally a wash solution cancomprise a detergent such as TWEEN-20 (e.g., about 0.01% to about 0.1%TWEEN-20).

In further aspects, methods of the embodiments concern detecting agalactose-containing containing molecule on a complex (i.e., a complexcomprising an antibody and a galactose-containing molecule) with adetectable lectin that binds galactose. For example, detecting agalactose-containing molecule can comprise (i) contacting the complexwith a lectin that binds galactose and (ii) detecting the bound lectin.In certain aspects, the method further comprises (i) contacting thecomplex with a lectin that binds galactose; (ii) washing the complex 1,2, 3, 4 or more times with a wash solution; and (iii) detecting thebound lectin. Examples of lectins for use according to the embodimentsinclude, without limitation mammalian galectin-3, Ricinus communislectin, Datura stramonium lectin, Erythrina cristagalli lectin, orLycopersicon esculentum lectin. In some aspects, the lectin is labeledwith a detectable label. In still further aspects, the lectin can bedetected with a labeled lectin-binding moiety (e.g., a labeledlectin-binding antibody).

The skilled artisan will recognize that the methods for detecting alectin will depend on the type of label that is employed. For example,in some aspects, a lectin comprises an affinity label (e.g., biotin) andthe label is itself detected by the binding of a further molecule ormolecules linked to a reporter (e.g., avidin linked to a reporter; insome alternatives, streptavidin can be used in place of avidin). Infurther aspects, a lectin may itself be labeled with a reporter.Reporter molecules for use according to the embodiments include, withoutlimitation, dyes, fluorophores, radionuclides and enzymes. For example,in certain aspects, the reporter is an enzyme, such a peroxidase, thatamplifies the detection signal by virtue of its catalytic activity. Avariety of enzymes can be used, such as an enzyme is selected from thegroup consisting of urease, alkaline phosphatase, horseradishperoxidase, glucose 6-phosphate dehydrogenase, β-galactosidase, andglucose oxidase.

Some aspects of the embodiments concern detecting a colorectal cancer bydetecting an increased level of a galactose-containing molecule. Incertain aspects, detecting an increased level of thegalactose-containing molecule comprises detecting a relative or absoluteincrease the level of the molecule. Further aspects of the embodimentsconcern quantifying a galactose-containing molecule in a serum sample.For example, a galactose-containing molecule can be quantified bycomparing an amount of lectin binding to a known reference sample or apanel of reference samples having known concentration agalactose-containing molecule (e.g., samples with known concentrationsof asialohaptoglobin). In certain aspects, such reference samples areassayed simultaneously with the serum sample. However, in some aspects,the reference sample values may be comprised in a reference table.

In further aspects a method of the embodiments further comprisesidentifying a subject as having a colorectal cancer or at risk of havinga colorectal cancer if the subject is determined to have an elevatedlevel of a galactose-containing molecule as compared with a controllevel. In certain aspects, identifying the subject further comprisesreporting that the subject has a colorectal cancer; is at risk of havinga colorectal cancer or has an elevated level of a 40-kDagalactose-containing molecule. For example, the reporting can compriseproviding a written, oral or electronic report. In some aspects, areport is provided to the tested subject, to a health care provider(e.g., a doctor), or to an insurance company.

In yet a further embodiment there is provided a method for monitoringthe effectiveness of an anti-cancer therapy comprising determining thelevel of a galactose-containing molecule (i.e., a molecule that binds toan anti-haptoglobin antibody) in a sample from a subject treated with ananti-cancer therapy in accordance with the embodiments. In some aspects,if the levels of the galactose-containing molecule are elevated ascompared to a reference level the subject is in need of additionalanti-cancer therapy. Conversely, if the level the galactose-containingmolecule not elevated as compared to a reference level the subject isnot in need of additional anti-cancer therapy. Thus, in certain aspects,a method is provided for monitoring the effectiveness of an anti-cancertherapy comprising (a) determining the levels of a galactose-containingmolecule in a sample from a subject before and after treatment with ananti-cancer therapy; and (b) identifying the subject as responsive tothe therapy or not responsive to the therapy based on the change inlevels of the galactose-containing molecule. For example, the level of agalactose-containing molecule in a responsive patient should be reducedafter therapy. In further aspects, a patient identified as notresponsive to the first anticancer therapy can be administered a secondanti-cancer therapy.

In some embodiments there is provided a method of treating a subjectcomprising administering an anticancer therapy to a subject identifiedas a having a colorectal cancer by a method of the embodiments. Forexample, the anticancer therapy can be a surgical, radiation,chemotherapeutic, or targeted anticancer therapy. In some aspects, asubject identified as having a pre-cancer can be treated by anendoscopic treatment of adenomatous polyps. In further aspects, asubject is treated to remove malignant polyps at colonoscopy.

In some aspects, a treatment of the embodiments comprises administeringan antimetabolites, such as capecitabine or fluorouracil; atopoisomerase inhibitor, such as irinotecan; a platinum-containingDNA-damaging agent, such as oxaliplatin; a dihydropyrimidinedehydrogenase inhibitor, such as tegafur/uracil; and/or an agent thatreduces toxicity when 5-fluorouracil is administered, such as folinicacid. Additional anticancer therapies include, but are not limited to,PD-1 receptor blockers, such as pembrolizumab or nivolumab. In someaspects, an anti-cancer agent is a monoclonal antibody or an anti-sensenucleic acid.

In still a further embodiment, there is provided a kit comprising atleast a first haptoglobin-binding antibody (e.g., the H-8636 antiserum)and a galactose-binding lectin (e.g., Erythrina cristagalli lectin). Incertain aspects, the antibody and/or the lectin is bound to a support orto a detectable label. In a further aspect, a kit of the embodimentscomprises a desialylating reagent such as a neuraminidase enzyme or amild acid, such as H₂SO₄. Further reagents that can be included in a kitof the embodiments include, without limitation, a microtiter plate, adetectable label, a lectin-binding antibody, and a dilution buffer(e.g., PBS or water).

In one alternative, the method further comprises the step of using ageas a co-variate variable to increase the sensitivity and specificity ofthe method.

In one alternative, the quantitation of step (4) is digitally scannedand uploaded to the server of a medical provider.

Another aspect of the invention is a method for detection and/ordiagnosis of colorectal cancer in a subject comprising:

-   -   (1) obtaining a serum sample from the subject;    -   (2) desialylating the serum sample;    -   (3) contacting the desialylated serum sample with an antibody        that binds haptoglobin to form a complex;    -   (4) quantitating the complex with a detectable lectin that binds        galactose in order to detect or diagnose colorectal cancer using        a quantitative immune-detection assay; and    -   (5) assaying the sample for the quantity of at least one        additional marker to confirm the detection or diagnosis of        colorectal cancer using one or more additional quantitative        immune-detection assays.

Typically, the at least one additional marker is at least one ofgalectin-3, a peptide derived from galectin-3, carcinoembryonic antigen(CEA), and CYFRA21-1.

In one alternative, the at least one additional marker is galectin-3,CYFRA21-1, and CEA. In another alternative, the at least one additionalmarker is CYFRA21-1 and CEA. In yet another alternative, the at leastone additional marker is CYFRA21-1. In still another alternative, the atleast one additional marker is CEA.

Typically, the one or more additional quantitative immune-detectionassays for assay of the quantity of the at least one additional markerare selected from the group consisting of an ELISA assay, aradioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay,a chemiluminescent immunoassay, a bioluminescent immunoassay, an enzymemultiplied immunoassay (EMIT), a cloned enzyme donor immunoassay(CEDIA), an immuno-PCR assay, a phosphor immunoassay, a quantum dotimmunoassay, a solid phase light-scattering immunoassay, a surfaceeffect immunoassay, and an immunoassay employing lateral flow teststrips. Preferably, the one or more additional quantitativeimmune-detection assays for assay of the quantity of the at least oneadditional marker are ELISA assays, such as sandwich assays. In certainaspects, an assay of the embodiments employs lateral flow test strips.Such lateral flow test strips for detection of analytes are described,for example, in United States Patent Application Publication No.2018/0356413; United States Patent Application Publication No.2014/0227725; PCT Patent Application Publication No. 2013/0132338; andPCT Patent Application Publication No. 2013/0132347, each of which isincorporated herein by reference. Such test strips can be employed inboth sandwich assays and competitive assays.

The method can further comprise the step of using age as a co-variatevariable to increase the sensitivity and specificity of the method.

In another alternative, the quantitation of step (d) is digitallyscanned and uploaded to the server of a medical provider.

In this alternative, the colorectal cancer can be selected from thegroup consisting of stage I colorectal cancer, stage II colorectalcancer, stage IIIA colorectal cancer, stage IIIB colorectal cancer,stage IIIC colorectal cancer, and stage IV colorectal cancer.

Another aspect of the invention is a method for detection and/ordiagnosis of a precancerous condition of the digestive tract in asubject comprising:

-   -   (1) obtaining a serum sample from the subject;    -   (2) desialylating the serum sample;    -   (3) contacting the desialylated serum sample with an antibody        that binds haptoglobin to form a complex; and    -   (4) quantitating the complex with a detectable lectin that binds        galactose in order to detect or diagnose the precancerous        condition of the digestive tract using a quantitative        immune-detection assay, wherein the presence of a        galactose-containing haptoglobin glycoform is associated with        the presence of a precancerous condition of the digestive tract,        wherein the quantitative immune-detection assay is not a Western        blot.

Typically, the precancerous condition of the digestive tract is anadenomatous polyp, such as an advanced adenoma.

In this method, the serum is desialylated as described above.Alternatives for the quantitative immune-detection assay are also asdescribed above. Suitable antibodies for the assay are also as describedabove. Other steps for the assay are also as described above. Typically,the detectable lectin that binds galactose is selected from the groupconsisting of Ricinus communis lectin, Sophora japonica lectin, Daturastramonium lectin, Erythrina cristagalli lectin, and Lycopersiconesculentum lectin; preferably, the detectable lectin is Erythrinacristagalli lectin. Methods for quantitating the complex formed in thecourse of the assay are also as described above. Typically, the reporteris an enzyme as described above.

The method can further comprise the step of using age as a co-variatevariable to increase the sensitivity and specificity of the method. Inone alternative, the quantitation of step (4) is digitally scanned anduploaded to the server of a medical provider.

The method can further comprise performance of a polypectomy to treat anadenomatous polyp.

Another aspect of the invention is a method for detection and/ordiagnosis of a precancerous condition of the digestive tract in asubject comprising:

-   -   (1) obtaining a serum sample from the subject;    -   (2) desialylating the serum sample;    -   (3) contacting the desialylated serum sample with an antibody        that binds haptoglobin to form a complex;    -   (4) quantitating the complex with a detectable lectin that binds        galactose in order to detect or diagnose a precancerous        condition of the digestive tract using a quantitative        immune-detection assay; and    -   (5) assaying the sample for the quantity of at least one        additional marker to confirm the detection or diagnosis of a        precancerous condition of the digestive tract using one or more        additional quantitative immune-detection assays.

Assay methods for this alternative, including the additional marker ormarkers, are as described above.

In this alternative, the method can further comprise performance of apolypectomy to treat an adenomatous polyp.

In one embodiment, there is provided a method for detecting colorectalcancer or a precancerous condition of the digestive tract in a subjectcomprising:

(a) measuring the level of galectin-3 ligand and CYFRA21-1 in a serumsample from the subject; and

(b) determining whether the subject has a colorectal cancer or acolorectal pre-cancerous lesion based on the level of galectin-3 ligandand the level of CYFRA21-1.

In some aspects, the method further comprises:

(a) measuring the level of galectin-3 ligand and CYFRA21-1 in a serumsample from the subject; and

(b) determining whether the subject has a colorectal cancer or acolorectal pre-cancerous lesion based on the level of galectin-3 ligand,the level of CYFRA21-1 and the age of the subject.

In some aspects, (a) further comprises measuring the level of oneadditional marker in the blood sample selected from the group consistingof galectin-3, a peptide derived from galectin-3 and carcinoembryonicantigen (CEA); and (b) further comprises determining whether the subjecthas a colorectal cancer or a precancerous condition of the digestivetract based on the level of said one additional marker.

In some aspects, (a) further comprises measuring the level of twoadditional markers in the blood sample selected from the groupconsisting of galectin-3, a peptide derived from galectin-3 andcarcinoembryonic antigen (CEA); and (b) further comprises determiningwhether the subject has a colorectal cancer or a precancerous conditionof the digestive tract based on the level of said two additionalmarkers.

In some aspects, the method further comprises:

(a) measuring the level of galectin-3 ligand, CYFRA21-1, galectin-3 andCEA in a blood sample from the subject; and

(b) determining whether the subject has a colorectal cancer or aprecancerous condition of the digestive tract based on the level ofgalectin-3 ligand, CYFRA21-1, galectin-3, CEA and the age of thesubject.

In some aspects, the blood sample is serum sample. In particularaspects, the method further comprises collecting the blood sample fromthe subject.

In some aspects, the method is used for determining whether the subjecthas a colorectal cancer. In specific aspects, the colorectal cancer isselected from the group consisting of stage I colorectal cancer, stageII colorectal cancer, stage IIIA colorectal cancer, stage IIIBcolorectal cancer, stage IIIC colorectal cancer, and stage IV colorectalcancer.

In some aspects, the method is a method for determining whether thesubject has a precancerous condition of the digestive tract. In someaspects, the precancerous condition of the digestive tract is anadenomatous polyp. In some aspects, the adenomatous polyp is an advancedadenoma.

In some aspects, measuring a level of galectin-3 ligand comprises:

-   -   (i) desialylating the blood sample;    -   (ii) contacting the desialylated sample with an antibody that        binds haptoglobin to form a complex; and    -   (iii) quantitating the complex with a detectable lectin that        binds galactose in order to measure the level of galectin-3        ligand in the sample.

In particular aspects, the sample is diluted between about 25-fold toabout 50-fold prior to desialylation. In specific aspects, the sample isdiluted at least 50-fold prior to desialylation. In some aspects, thedesialylated sample is further diluted to a final dilution of betweenabout 100-fold and 150,000-fold; of between about 1,000-fold and about150,000-fold, or about 15,000-fold and about 150,000-fold before step(ii). In a further aspect, the desialylated sample is diluted to a finaldilution of about 20,000-fold before step (ii).

In some aspects, quantitating the complex is done by a quantitativeimmune-detection assay. In particular aspects, the quantitativeimmune-detection assay is not a Western blot. In some aspects, thequantitative immune-detection assay is . an ELISA assay, aradioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay,a chemiluminescent immunoassay, a bioluminescent immunoassay, an enzymemultiplied immunoassay (EMIT), a cloned enzyme donor immunoassay(CEDIA), an immuno-PCR assay, a phosphor immunoassay, a quantum dotimmunoassay, a solid phase light-scattering immunoassay, a surfaceeffect immunoassay, or an immunoassay employing lateral flow teststrips. In specific aspects, the quantitative immune-detection assay isan ELISA. In a particular aspect, the ELISA is a sandwich ELISA.

In some aspects, desialylating the sample comprises treating the bloodsample with a mild acid. In some aspects, the mild acid is H₂SO_(4.) Insome aspects, desialylating the sample comprises treating the samplewith a neuraminidase.

In some aspects, the antibody that binds haptoglobin is a polyclonalantibody. In particular aspects, the antibody that binds haptoglobin isbound to a substrate or a magnetic bead. In specific aspects, theantibody that binds haptoglobin is a polyclonal antibody raised againstpurified human haptoglobin.

In some aspects, the method further comprises a step of contacting thecomplex of step (ii) with a wash solution prior to step (ii),In someaspects, the wash solution comprises a detergent.

In some aspects, the detectable lectin that binds galactose is selectedfrom the group consisting of Ricinus communis lectin, Sophora japonicalectin, Datura stramonium lectin, Erythrina cristagalli lectin, andLycopersicon esculentum lectin. In particular aspects, the detectablelectin is Erythrina cristagalli lectin.

In some aspects, quantitating the complex with a detectable lectincomprises detecting an enzymatic activity. In some aspects, the lectinis biotinylated or comprises a conjugated enzyme. In particular aspects,the lectin is biotinylated and detecting the complex comprisescontacting the complex with an avidin-reporter conjugate.

In some aspects, the measuring is automated. In some aspects, measuringcomprises performing a quantitative immune-detection assay. Inparticular aspects, the quantitative immune-detection assay is an ELISAassay, a radioimmunoassay (RIA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescentimmunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzymedonor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay,a quantum dot immunoassay, a solid phase light-scattering immunoassay, asurface effect immunoassay or an immunoassay employing lateral flow teststrips. In specific aspects, the quantitative immune-detection assays isan ELISA assay, such as a sandwich ELISA assay. In some aspects, theELISA assay is sandwich ELISA assay.

In another embodiment, there is provided a method of treating a subjectcomprising administering a colonoscopy to a subject determined to have acolorectal pre-cancerous lesion in accordance with any of aforementionedembodiments and aspects thereof.

In another embodiment, there is provided a method of treating a subjectcomprising performance of a polypectomy on a subject determined to havea precancerous condition of the digestive tract in accordance with anyof the aforementioned embodiments or aspects thereof. In some aspects,the precancerous condition of the digestive tract is an adenomatouspolyp.

In another embodiment, there is provided a method of treating a subjectcomprising administering an anti-cancer therapy to a subject determinedto have a colorectal cancer or a precancerous condition of the digestivetract in accordance with any of the aforementioned embodiments oraspects thereof. In some aspects, the subject was determined to have acolorectal cancer. In some aspects, the colorectal cancer is selectedfrom the group consisting of stage I colorectal cancer, stage IIcolorectal cancer, stage IIIA colorectal cancer, stage IIIB colorectalcancer, stage IIIC colorectal cancer, and stage IV colorectal cancer.

In another embodiment, there is provided a method for detectingcolorectal cancer or colorectal pre-cancerous lesions in a subjectcomprising:

-   -   (a) measuring the level of galectin-3 ligand in a blood sample        from the subject; and    -   (b) determining whether the subject has a colorectal cancer or a        precancerous condition of the digestive tract based on the level        of galectin-3 ligand and the age of the subject.

In some aspects, the method further comprises:

(a) measuring the level of galectin-3 ligand and CYFRA21-1 in a bloodsample from the subject; and (b) determining whether the subject has acolorectal cancer or a precancerous condition of the digestive tractbased on the level of galectin-3 ligand, the level of CYFRA21-1 and theage of the subject.

In some aspects, (a) further comprises measuring the level of oneadditional marker in the blood sample selected from the group consistingof galectin-3, a peptide derived from galectin-3 and carcinoembryonicantigen (CEA); and (b) further comprises determining whether the subjecthas a colorectal cancer or a precancerous condition of the digestivetract based on the level of said one additional marker.

In some aspects, (a) further comprises measuring the level of twoadditional markers in the blood sample selected from the groupconsisting of galectin-3, a peptide derived from galectin-3 andcarcinoembryonic antigen (CEA); and (b) further comprises determiningwhether the subject has a colorectal cancer or a precancerous conditionof the digestive tract based on the level of said two additionalmarkers.

In some aspects, the method further comprises:

(a) measuring the level of galectin-3 ligand, CYFRA21-1, galectin-3 andCEA in a serum sample from the subject; and

(b) determining whether the subject has a colorectal cancer or aprecancerous condition of the digestive tract based on the level ofgalectin-3 ligand, CYFRA21-1, galectin-3, CEA and the age of thesubject.

In some aspects, the blood sample is serum sample. In particularaspects, the method further comprises collecting the blood sample fromthe subject.

In some aspects, the method is used for determining whether the subjecthas a colorectal cancer. In specific aspects, the colorectal cancer isselected from the group consisting of stage I colorectal cancer, stageII colorectal cancer, stage IIIA colorectal cancer, stage IIIBcolorectal cancer, stage IIIC colorectal cancer, and stage IV colorectalcancer.

In some aspects, the method is a method for determining whether thesubject has a precancerous condition of the digestive tract. In someaspects, the precancerous condition of the digestive tract is anadenomatous polyp. In some aspects, the adenomatous polyp is an advancedadenoma.

In some aspects, measuring a level of galectin-3 ligand comprises:

-   -   (i) desialylating the blood sample;    -   (ii) contacting the desialylated sample with an antibody that        binds haptoglobin to form a complex; and    -   (iii) quantitating the complex with a detectable lectin that        binds galactose in order to measure the level of galectin-3        ligand in the sample.

In particular aspects, the sample is diluted between about 25-fold toabout 50-fold prior to desialylation. In specific aspects, the sample isdiluted at least 50-fold prior to desialylation. In some aspects, thedesialylated sample is further diluted to a final dilution of betweenabout 100-fold and 150,000-fold; of between about 1,000-fold and about150,000-fold, or about 15,000-fold and about 150,000-fold before step(ii). In a further aspect, the desialylated sample is diluted to a finaldilution of about 20,000-fold before step (ii).

In some aspects, quantitating the complex is done by a quantitativeimmune-detection assay. In particular aspects, the quantitativeimmune-detection assay is not a Western blot. In some aspects, thequantitative immune-detection assay is . an ELISA assay, aradioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay,a chemiluminescent immunoassay, a bioluminescent immunoassay, an enzymemultiplied immunoassay (EMIT), a cloned enzyme donor immunoassay(CEDIA), an immuno-PCR assay, a phosphor immunoassay, a quantum dotimmunoassay, a solid phase light-scattering immunoassay, a surfaceeffect immunoassay, or an immunoassay employing lateral flow teststrips. In specific aspects, the quantitative immune-detection assay isan ELISA. In a particular aspect, the ELISA is a sandwich ELISA.

In some aspects, desialylating the sample comprises treating the bloodsample with a mild acid. In some aspects, the mild acid is H₂SO₄. Insome aspects, desialylating the sample comprises treating the samplewith a neuraminidase.

In some aspects, the antibody that binds haptoglobin is a polyclonalantibody. In particular aspects, the antibody that binds haptoglobin isbound to a substrate or a magnetic bead. In specific aspects, theantibody that binds haptoglobin is a polyclonal antibody raised againstpurified human haptoglobin.

In some aspects, the method further comprises a step of contacting thecomplex of step (ii) with a wash solution prior to step (ii). In someaspects, the wash solution comprises a detergent.

In some aspects, the detectable lectin that binds galactose is selectedfrom the group consisting of Ricinus communis lectin, Sophora japonicalectin, Datura stramonium lectin, Erythrina cristagalli lectin, andLycopersicon esculentum lectin. In particular aspects, the detectablelectin is Erythrina cristagalli lectin.

In some aspects, quantitating the complex with a detectable lectincomprises detecting an enzymatic activity. In some aspects, the lectinis biotinylated or comprises a conjugated enzyme. In particular aspects,the lectin is biotinylated and detecting the complex comprisescontacting the complex with an avidin-reporter conjugate.

In some aspects, the measuring is automated. In some aspects, measuringcomprises performing a quantitative immune-detection assay. Inparticular aspects, the quantitative immune-detection assay is an ELISAassay, a radioimmunoassay (RIA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescentimmunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzymedonor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay,a quantum dot immunoassay, a solid phase light-scattering immunoassay, asurface effect immunoassay or an immunoassay employing lateral flow teststrips. In specific aspects, the quantitative immune-detection assays isan ELISA assay, such as a sandwich ELISA assay. In some aspects, theELISA assay is sandwich ELISA assay.

In still further aspects, a method of the embodiments comprises the useof europium chelate particles as labels. For example, europiumchelate-loaded silica nanoparticles can be used as a label for detectionby fluorescence (see, e.g., X. Xia et al., “Lateral Flow ImmunoassayUsing Europium Chelate-Loaded Silica Nanoparticles as Labels,” Clin.Chem. 55: 179-182 (2009), incorporated herein by reference). In someaspects, the europium is not radioactive.

In another embodiment, there is provided a method of treating a subjectcomprising administering a colonoscopy to a subject determined to have acolorectal cancer or a precancerous condition of the digestive tract inaccordance with any of aforementioned embodiments and aspects thereof.

In another embodiment, there is provided a method of treating a subjectcomprising performance of a polypectomy on a subject determined to havea precancerous condition of the digestive tract in accordance with anyof the aforementioned embodiments or aspects thereof. In some aspects,the precancerous condition of the digestive tract is an adenomatouspolyp.

In another embodiment, there is provided a method of treating a subjectcomprising administering an anti-cancer therapy to a subject determinedto have a colorectal cancer condition of the digestive tract inaccordance with any of the aforementioned embodiments or aspectsthereof. In some aspects, the subject was determined to have acolorectal cancer. In some aspects, the colorectal cancer is selectedfrom the group consisting of stage I colorectal cancer, stage IIcolorectal cancer, stage IIIA colorectal cancer, stage IIIB colorectalcancer, stage IIIC colorectal cancer, and stage IV colorectal cancer.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein. Likewise, aspects of the present embodiments discussed in thecontext of a method quantifying are equally applicable to any method fordiagnosing or detecting according to the embodiments.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

An “anti-cancer” agent or therapy is capable of negatively affecting acancer cell/tumor in a subject, for example, by promoting killing ofcancer cells, inducing apoptosis in cancer cells, reducing the growthrate of cancer cells, reducing the incidence or number of metastases,reducing tumor size, inhibiting tumor growth, reducing the blood supplyto a tumor or cancer cells, promoting an immune response against cancercells or a tumor, preventing or inhibiting the progression of cancer, orincreasing the lifespan of a subject with cancer.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B: (A) Sandwich ELISA of asialohaptoglobin prepared byneuraminidase vs. mild acid hydrolysis at 1.25× dilution. (B) SandwichELISA of asialohaptoglobin prepared by neuraminidase vs. mild acidhydrolysis at 5× dilution.

FIG. 2: Comparison of assay formats. Known serum samples, desialylatedby mild acid hydrolysis at 5× dilution, were assayed with Erythrinacristagalli lectin (ECL) catcher and anti-haptoglobin tracer or withanti-haptoglobin tracer and biotinyl-ECL tracer. Neuraminidase-treatedserum samples were assayed in parallel.

FIG. 3: Constructed receiver operating characteristic curves ofsensitivity versus (1-specificity) using a sandwich ELISA of theembodiments. Results demonstrated that the assay successfullydifferentiated individuals with colorectal neoplasia from normalcontrols with a high degree of sensitivity and specificity. The AUC forthe 40-kDa haptoglobin glycoform (galectin-3 (Ga13) ligand) alone,normal versus cancer, was 0.84 (left panel) and for Ga13 ligand+fecaloccult blood test (FOBT) was 0.91 (right panel).

FIG. 4: Quantitative ELISA detection of the 40-kDa haptoglobin glycoformis highly reproducible. Graphs show assay of serum samples assayed on 3separate plates (with serum order sequential, shuffled, or scrambled) onseparate days (3 sets of 11 plates each in batches of 3 or 4 plates).Net A₄₀₅ values were used to calculate mg/ml for each assay set(y-axis), which is compared to the median mg/ml for each serum (x-axis).

FIG. 5: Graphs show assay of serum samples of separate days (A). Eachplate had triplicate reference standard of 20,000-fold diluted mildacid-treated colon cancer serum 10 (EDRN #73249037, local name C5) andnormal serum (EDRN #73265650, local name N4). B shows the mean of theassay in A.

FIG. 6: Protein concentrations as assessed by a method of theembodiments from samples assayed in 2009 (x-axis) versus 2012 (y-axis).

FIG. 7: Constructed receiver operating characteristic (ROC) curves ofsensitivity versus (1-specificity) using a bead-based assay of theembodiments.

FIGS. 8A-8D: ROC curves for galectin-3 ligand alone for differentiatingnormal individuals without cancer from other groups: (A) advancedadenoma; (B) early stage cancer (stages I and II); (C) late stage cancer(stages III and IV); and (D) all cancers.

FIG. 9: Performance of marker panels by patient group showing AUC withsensitivities at 85% and 90% specificities; the patient groups versusnormal are: (i) all colorectal cancer (CRC); (ii) advanced adenoma;(iii) stage I and II CRC; (iv) stage I, II, IIIA, and IIIB CRC; (iv)stage IIIC and IV CRC; and (v) SRN (all CRC and advanced adenoma). Themodels shown are: (i) galectin-3 ligand (Model 0); (ii) galectin-3ligand, CEA, CYFRA21-1, and galectin-3 (Model 1); (iii) galectin-3ligand, CEA, and CYFRA21-1 (Model 2); (iv) galectin-3 ligand andCYFRA21-1 (Model 3); and (v) galectin-3 ligand and CEA (Model 4).

FIGS. 10A-10F: ROC curves for: Models 1, 2, 3, and 4: (A) for allcancers vs. normal; (B) advanced adenoma vs. normal; (C) stage I and IIof colorectal cancer vs. normal; (D) stage I, II, and III of colorectalcancer vs. normal; (E) stage IV of colorectal cancer vs. normal; and (F)all colorectal cancers and advanced adenoma.

FIG. 11: ROC curves for Model 2 for all cancers vs. normal using datafrom Danish Endoscopy II trial specimens.

FIG. 12A-12F: ROC curves for use of age as a co-variate together withgalectin-3 ligand (solid lines) as compared with galectin-3 ligand andage as a co-variate (dashed lines). (A) shows small adenomas vs. normalswithout comorbidity. (B) shows advanced adenomas vs. normals withoutcomorbidity. (C) shows all adenomas vs. normals without comorbidity. (D)shows early cancer vs. normals without comorbidity. (E) shows latecancer vs. normals without comorbidity. (F) shows all cancer vs. normalswithout comorbidity.

FIG. 13: is a schematic diagram of the reaction as performed on a bead.Step 1 is the coupling reaction in which an anti-haptoglobin antibody iscoupled to a bead. Step 2 is the binding of haptoglobin from serum withand without the galectin-3 ligand to the anti-haptoglobin antibodycoupled to the bead. Step 3 is the secondary binding of a biotinylatedlectin vector to the haptoglobin; only the haptoglobin with thegalectin-3 ligand binds to the lectin at this stage. Step 4 is thebinding of a streptavidin-phycoerythrin conjugate to the bead; thestreptavidin binds only to the biotinylated lectin vector bound to thehaptoglobin.

FIG. 14: is a table showing the performance sensitivity and specificityof a bead assay for galectin-3 ligand, with breakdown of the subjects asnormal, small adenoma, advanced adenoma, cancer at Stage III, cancer atStage IIIA/B, or cancer at Stage IIIC/IV.

FIG. 15: is a graphical representation of the data from FIG. 14.

FIG. 16: is a table showing the performance of plate and bead assays forgalectin-3 ligand for high-risk adenomas, cancer at Stage III, cancer atStage III/IV, or all cancers.

FIG. 17: is a table showing the performance of individual cancer markersby patient group (advanced adenoma, early colorectal cancer, latecolorectal cancer, all colorectal cancer, and colorectal cancer plusadvanced adenoma). The individual cancer markers analyzed are galectin-3ligand (i.e., the haptoglobin glycoform), carcinoembryonic antigen(CEA), galectin-3, MAPRE-1, ferritin, CYFRA21-1, and CRP16.

FIG. 18: is a table showing a comparison of the results with thegalectin-3 ligand test in the bead format with and without age as acovariate for small adenomas, advanced adenomas, all adenomas, cancerStage I/II, cancer Stage III/IV, and all cancers.

FIG. 19: are strips showing haptoglobin standards and serum samplesdetected by Europium chelate nanoparticles. Based on the standard curve,a DC/DN ratio of 6.79 was observed indicating the LFA is successful atdiscriminating between the cancer and normal samples.

FIG. 20: is a table showing galectin-3 ligand values versus number ofsamples.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The quality of any diagnostic method, such as a method that attempts todetermine the presence or absence of colorectal cancer, is determined bythe proportion of false negative and false positive results obtained.The ideal, which is not attainable in practice, is to have a falsenegative rate and a false positive rate of zero; in that case, everypatient screened in which the method indicates the presence ofcolorectal cancer actually has the disease, while every patient screenedin which the method indicates the absence of colorectal cancer is freeof the disease when the test was performed. A false negative result is atest result in which a patient screened by the test and in which thetest indicates that the patient is free of the disease actually has thedisease, colorectal cancer in this instance. A false positive result isa test result in which a patient screened by the test and in which thetest indicates that the patient has the disease actually is free of thedisease. The sensitivity of the test is the proportion of patients whotest positive for the disease among those who actually have the disease;the higher the sensitivity, the lower the proportion of false negativeresults. The specificity of the test is the proportion of patients whotest negative for the disease among those who actually are free of thedisease; the higher the specificity, the lower the proportion of falsepositive results. The consequences of false positive and false negativeresults with respect to colorectal cancer are significantly different. Afalse positive result, although it can have temporary psychologicalconsequences for the patient receiving the false positive result, can becorrected by rescreening by another test method, and, once the absenceof colorectal cancer is confirmed by additional tests, there are nosignificant long-term consequences, although, in some cases, if thoseadditional tests are themselves invasive, there may be a risk factorassociated with the administration of the tests. A false negative resultin a patient in which colorectal cancer exists, on the other hand, canhave more serious consequences, as an undiagnosed malignancy willcontinue to grow without treatment and may be untreatable or may requiretreatment with a lower probability of success or more serious sideeffects by the time the malignancy is actually diagnosed. Therefore, itis the goal of any diagnostic test for any form of cancer, includingcolorectal cancer, to reduce false negatives as much as is practicable,while keeping the number of false positives to an acceptable level.

Embodiments of the invention provide a sensitive and reproducible assayfor the detection of colorectal cancer. In particular, the inventorshave identified a detectable form of a glycoprotein (40-kDa) that bindsto anti-haptoglobin antibodies, but is distinct from other humanhaptoglobin glycoforms, and that is elevated specifically in patientswith colorectal cancer. Because the protein appears to be present atsome level in healthy patients and is immunologically related tohaptoglobin, the inventors have developed techniques that allow forquantitative detection only of specific glycosylated forms of the 40-kDaprotein. The assay involves obtaining a diluted serum sample frompatient and treating the bulk sample to desialylate proteins that arepresent. The desialylated and diluted serum sample is then subjected toa highly sensitive sandwich ELISA assay by capturing the 40-kDa proteinusing an anti-haptoglobin antibody and detecting the captured proteinwith a detectable lectin that binds to galactose. By using adesialylated serum sample that has been diluted to between 100-fold and50,000-fold the assay allows for the amount of the 40-kDa glycoform in asample to be determined quantitatively. An increased level of the 40-kDaglycoform (e.g., as compared to a control sample or a reference level)is indicative of the presence of colorectal cancer in the patient.

The assay systems and methods provided offer significant advantages overconventional methods for detecting colorectal cancer. For example,whereas a colonoscopy is typically only performed once every 5-10 yearsdue to its cost and invasive nature, the blood tests of the embodimentscould be repeated as often as desired by a physician, such as everyyear, twice a year or even every month. Moreover, such small samplevolumes are required for the tests detailed here that portions of bloodtaken for other purposes could be tasked for use in the assay. Theability to repeat these tests frequently and their exquisite sensitivityoffer the opportunity to detect cancers at a very early stage and thusto apply therapy at a time when it can be most effective in improvingpatient outcome.

I. IMMUNOLOGICAL REAGENTS

In certain aspects of the invention, one or more antibodies are employedthat bind to the 40-kDa haptoglobin glycoform. Antibodies include anytype of antibody, and specifically refer to antibodies that reactimmunologically with human haptoglobin or immunologically relatedproteins such as the 40-kDa haptoglobin glycoform. In particular, theseantibodies may be used in various diagnostic applications, such as theELISA assay methods detailed below.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. The term“antibody” is used to refer to any antibody-like molecule that has anantigen binding region, and includes antibody fragments such as Fab',Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (single chainFv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (see, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

Antibodies can be polyclonal or monoclonal. Monoclonal antibodies (MAbs)are recognized to have certain advantages, e.g., reproducibility andlarge-scale production. The invention thus provides monoclonalantibodies of the human, murine, monkey, rat, hamster, rabbit and evenchicken origin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be utilized.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal with,for example a human haptoglobin (e.g., a purified haptoglobin)composition in accordance with the present invention and collectingantisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically, the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon according to the ease of manipulation, costsor the desired amount of sera, as would be known to one of skill in theart.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia or syntheticcompositions.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,interferon, GM-CSF, CSF, BCG, aluminum hydroxide, MDP compounds, such asnor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI,which contains three components extracted from bacteria, MPL, trehalosedimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80emulsion is also contemplated. MHC antigens may even be used. Exemplaryadjuvants include complete Freund's adjuvant (a non-specific stimulatorof the immune response containing killed Mycobacterium tuberculosis),incomplete Freund' s adjuvant and aluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead, N.J.),cytokines such as α-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography. Thus, in certain aspects,polyclonal antibodies for used according to the embodiments are purifiedantibodies, such an IgG fraction of antibodies.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. In some embodiments, however, the antibody that reactsimmunologically with the anti-tumor antigen antibody and/or theanti-tumor antigen antibody are present endogenously in a subject.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are often used, however, the use ofrabbit, sheep or frog cells is also possible.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by 5 homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non-antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender them incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U, MPC 11,MPC11 X45 GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3, Y3Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICR LON HMy2 andUC729 6 are all useful in connection with human cell fusions.

One particular murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine resistant mouse murine myeloma SP2/0 non producer cellline.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKöhler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryagents are aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides (HAT medium). Where azaserine isused, the media is supplemented with hypoxanthine.

The favored selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 10⁴ times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length genes or gene fragments in Escherichiacoli.

II. LECTINS

It has long been known that extracts from certain plants couldagglutinate red blood cells. Although the term “lectin” was originally aterm used to describe agglutinins which could discriminate among typesof red blood cells, the term is now generally defined to includesugar-binding proteins from a wide variety of sources. Lectins have beenfound in plants, viruses, microorganisms and animals. Although lectinsshare the common property of binding to defined sugar structures, theirroles in various organisms are not likely to be the same and remainincompletely understood.

Because of the specificity that each lectin has toward a particularcarbohydrate structure, even oligosaccharides with identical sugarcompositions can be distinguished or separated. Some lectins will bindonly to structures with mannose or glucose residues, while others mayrecognize only galactose residues. Some lectins require that theparticular sugar be in a terminal non-reducing position in theoligosaccharide, while others can bind to sugars within theoligosaccharide chain. Some lectins do not discriminate between alphaand beta anomers, while others require not only the correct anomericstructure but a specific sequence of sugars for binding. The affinitybetween a lectin and its receptor may vary a great deal due to smallchanges in the carbohydrate structure of the receptor.

Thus, lectins can be used in similar detection methods as antibodies,for the detection of specific carbohydrate moieties. Embodiments of thepresent invention provide assays for the detection of a 40-kDahaptoglobin glycoform using lectins as selective binding agents.Generally, the carbohydrate composition of the 40-kDa glycoform isexploited in order to detect its presence in a sample. For example,embodiments of the methods of the invention employ galactose-bindinglectins to capture or detect the 40-kDa protein comprising such agalactose moiety.

In the study detailed here, the inventors used galactose binding lectinsto detect the 40-kDa glycoform in desialylating the diluted serum.Lectins that were found to be effective include mammalian galectin-3,Ricinus communis lectin, Datura stramonium lectin, Erythrina cristagallilectin, and Lycopersicon esculentum lectin. Of these however, thehighest specificity was achieved with Erythrina cristagalli lectin.

In some aspects, lectins for use according to the embodiments arelabeled. Methods for labeling antibodies can generally also be appliedto lectins and are further detailed below.

III. ANTIBODY AND LECTIN CONJUGATES

The present invention further provides antibodies and lectins reactivewith human haptoglobin and the 40-kDa haptoglobin glycoform that arelinked to at least one agent to form an antibody or lectin conjugate. Inorder to increase the efficacy of such conjugates as diagnostic agents,it is conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one reporter molecule. Any antibody or lectin ofsufficient selectivity, specificity or affinity may be employed as thebasis for a conjugate. Such properties may be evaluated usingconventional immunological screening methodology known to those of skillin the art. It will therefore be understood that embodiments referringto antibody conjugates are thus equally applicable to lectin conjugatesand vice versa.

B. Conjugation

Thus, in certain aspects a lectin or antibody of the embodiments isconjugated to a reporter. Any of a wide array of conjugation schemes canbe employed to provide linkage of an antibody or lectin to a reporter asfurther detailed below.

An exemplary heterobifunctional cross-linker contains two reactivegroups: one reacting with a primary amine group (e.g.,N-hydroxysuccinimide) and the other reacting with a thiol group (e.g.,pyridyl disulfide, maleimides, halogens, or other agents capable ofreacting with thiols). Through the primary amine reactive group, thecross-linker may react with the lysine residue(s) of one protein (e.g.,the selected antibody or lectin) and through the thiol reactive group,the cross-linker, already tied up to the first protein, reacts with thecysteine residue (free sulfhydryl group) of the other protein (e.g., thereporter). In some cases, it is preferred that a cross-linker havingreasonable stability in serum samples will be employed. Numerous typesof disulfide-bond containing linkers are known that can be successfullyemployed to conjugate targeting and therapeutic/preventative agents.These linkers are thus one group of linking agents.

Another cross-linking reagent is SMPT(4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene),which is a bifunctional cross-linker containing a disulfide bond that issterically hindered by an adjacent benzene ring and methyl groups. It isbelieved that steric hindrance of the disulfide bond serves a functionof protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to the delivery of theattached agent to the target site. SMPT provides amine-to-sulfhydrylconjugation via NHS-ester and pyridyldithiol reactive groups.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the ε-amino group of lysine).Another possible type of cross-linker includes the heterobifunctionalphotoreactive phenylazides containing a cleavable disulfide bond such assulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate.The N-hydroxy-succinimidyl group reacts with primary amino groups andthe phenylazide (upon photolysis) reacts non-selectively with any aminoacid residue.

In addition to hindered cross-linkers, non-hindered cross-linkers alsocan be employed in methods according to the present invention. Otheruseful cross-linkers, not considered to contain or generate a protecteddisulfide, include SATA (N-succinimidyl S-acetylthioacetate), SPDP(succinimidyl 3-(2-pyridylthio)propionate), and 2-iminothiolane(Wawrzynczak & Thorpe, 1988). The use of such cross-linkers is wellunderstood in the art. Another embodiment involves the use of flexiblelinkers.

U.S. Pat. No. 4,680,338 describes bifunctional linkers useful forproducing conjugates of ligands with amine-containing polymers and/orproteins, especially for forming antibody conjugates with chelators,drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648and 5,563,250 disclose cleavable conjugates containing a labile bondthat is cleavable under a variety of mild conditions. This linker isparticularly useful in that the agent of interest may be bonded directlyto the linker, with cleavage resulting in release of the active agent.Preferred uses include adding a free amino or free sulfhydryl group to aprotein, such as an antibody, or a drug.

U.S. Pat. No. 5,856,456 provides peptide linkers for use in connectingpolypeptide constituents to make fusion proteins, e.g., single chainantibodies. The linker is up to about 50 amino acids in length, containsat least one occurrence of a charged amino acid (preferably arginine orlysine) followed by a proline, and is characterized by greater stabilityand reduced aggregation. U.S. Pat. No. 5,880,270 disclosesaminooxy-containing linkers useful in a variety of immunodiagnostic andseparative techniques.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Some attachment methods involve the use of a metal chelate complexemploying, for example, an organic chelating agent such adiethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3,6-diphenylglycouril-3 attached to the antibody (U.S. Pat.Nos. 4,472,509 and 4,938,948, each incorporated herein by reference).Antibodies or lectins may also be reacted with an enzyme in the presenceof a coupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate. In U.S. Pat. No.4,938,948, imaging of breast tumors is achieved using monoclonalantibodies and the detectable imaging moieties are bound to the antibodyusing linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

Other cross-linking agents are known in the art. Suitable reagents forcross-linking many combinations of functional groups are known in theart. For example, electrophilic groups can react with many functionalgroups, including those present in proteins or polypeptides. Variouscombinations of reactive amino acids and electrophiles are known in theart and can be used. For example, N-terminal cysteines, containing thiolgroups, can be reacted with halogens or maleimides. Thiol groups areknown to have reactivity with a large number of coupling agents, such asalkyl halides, haloacetyl derivatives, maleimides, aziridines, acryloylderivatives, arylating agents such as aryl halides, and others. Theseare described in G. T. Hermanson, “Bioconjugate Techniques” (AcademicPress, San Diego, 1996), pp. 146-150. The reactivity of the cysteineresidues can be optimized by appropriate selection of the neighboringamino acid residues. For example, a histidine residue adjacent to thecysteine residue will increase the reactivity of the cysteine residue.Other combinations of reactive amino acids and electrophilic reagentsare known in the art. For example, maleimides can react with aminogroups, such as the ε-amino group of the side chain of lysine,particularly at higher pH ranges. Aryl halides can also react with suchamino groups. Haloacetyl derivatives can react with the imidazolyl sidechain nitrogens of histidine, the thioether group of the side chain ofmethionine, and the .epsilon.-amino group of the side chain of lysine.Many other electrophilic reagents are known that will react with theE-amino group of the side chain of lysine, including, but not limitedto, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimideesters, sulfonyl chlorides, epoxides, oxiranes, carbonates, imidoesters,carbodiimides, and anhydrides. These are described in G. T. Hermanson,“Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp.137-146. Additionally, electrophilic reagents are known that will reactwith carboxylate side chains such as those of aspartate and glutamate,such as diazoalkanes and diazoacetyl compounds, carbonydiimidazole, andcarbodiimides. These are described in G. T. Hermanson, “BioconjugateTechniques” (Academic Press, San Diego, 1996), pp. 152-154. Furthermore,electrophilic reagents are known that will react with hydroxyl groupssuch as those in the side chains of serine and threonine, includingreactive haloalkane derivatives. These are described in G. T. Hermanson,“Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp.154-158. In another alternative embodiment, the relative positions ofelectrophile and nucleophile (i.e., a molecule reactive with anelectrophile) are reversed so that the protein has an amino acid residuewith an electrophilic group that is reactive with a nucleophile and thetargeting molecule includes therein a nucleophilic group. This includesthe reaction of aldehydes (the electrophile) with hydroxylamine (thenucleophile), described above, but is more general than that reaction;other groups can be used as electrophile and nucleophile. Suitablegroups are well known in organic chemistry and need not be describedfurther in detail.

Additional combinations of reactive groups for cross-linking are knownin the art. For example, amino groups can be reacted withisothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS)esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes,carbonates, alkylating agents, imidoesters, carbodiimides, andanhydrides. Thiol groups can be reacted with haloacetyl or alkyl halidederivatives, maleimides, aziridines, acryloyl derivatives, acylatingagents, or other thiol groups by way of oxidation and the formation ofmixed disulfides. Carboxy groups can be reacted with diazoalkanes,diazoacetyl compounds, carbonyldiimidazole, carbodiimides. Hydroxylgroups can be reacted with epoxides, oxiranes, carbonyldiimidazole,N,N′-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate,periodate (for oxidation), alkyl halogens, or isocyanates. Aldehyde andketone groups can react with hydrazines, reagents forming Schiff bases,and other groups in reductive amination reactions or Mannichcondensation reactions. Still other reactions suitable for cross-linkingreactions are known in the art. Such cross-linking reagents andreactions are described in G. T. Hermanson, “Bioconjugate Techniques”(Academic Press, San Diego, 1996).

C. Reporter Molecules

A reporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules which have beenconjugated to antibodies or lectins include enzymes, radiolabels,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

In the case of radioactive isotopes that can be conjugated to antibodies(or lectins) for diagnostic applications, one might mention ²¹¹astatine,¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁷copper,¹⁵²europium, ⁶⁷gallium, ³hydrogen, ¹²³iodine,¹²⁵iodine, ¹³¹iodine,¹¹¹indium, ⁵⁹iron, ³²phosphorus, ¹⁸⁶rhenium, ¹⁸⁸rhenium, ⁷⁵selenium,³⁵sulfur, ⁹⁹mtechnetium, and/or ⁹⁰yttrium. Radioactively labeledantibodies of the present invention may be produced according towell-known methods in the art. For instance, antibodies or lectins canbe iodinated by contact with sodium and/or potassium iodide and achemical oxidizing agent such as sodium hypochlorite, or an enzymaticoxidizing agent, such as lactoperoxidase. Antibodies or lectinsaccording to the invention may be labeled with ^(99m)technetium by aligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SnCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of conjugate contemplated in the present invention is thosewhere the antibody or lectin is linked to a secondary binding ligandand/or to an enzyme (an enzyme tag) that will generate a colored productupon contact with a chromogenic substrate.

Examples of suitable enzymes include urease, alkaline phosphatase,horseradish peroxidase, glucose 6-phosphate dehydrogenase,β-galactosidase, and glucose oxidase. Secondary binding ligands arebiotin and/or avidin and streptavidin compounds. Streptavidin is a52.8-kDa protein purified from Streptomyces avidinii with an extremelyhigh affinity for biotin, with a dissociation constant (K_(d)) on theorder of about 10⁻¹⁴ mol/L. Streptavidin is an alternative to avidin.The use of such labels is well known to those of skill in the art and isdescribed, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; eachincorporated herein by reference.

Likewise, in certain applications it is desirable to immobilize anantibody on a solid surface. For example, antibodies can be absorbed tothe surface of the wells of a plate. Such absorption can rely onnon-specific interactions with the polymers of the plate or can involvespecific binding of the antibodies (e.g., by protein A). In certainaspects antibodies are covalently linked to a surface, for example byUV-cross linking.

Optical imaging with dyes also permit visualization of biologicalactivities (Blasdel et al., 1986; Grinvald et al., 1988; Kauer et al.,1988; Lieke et al., 1989). Dyes that are sensitive to physicochemicalenvironments (such as pressure, cell membrane potential, ionconcentration, acidity, partial pressure of oxygen, or otherenvironmental changes) are subject to changes in absorption or emissionof light. The resulting changes act as optical probes to transformbiological activities into optical signals that can be converted intooptical images.

Water soluble dyes are particularly well suited for optical imaging,including acid dyes, basic dyes, direct dyes, and so on, and equivalentsthereof. The dye composition may be prepared as a dry material for easeof storage and packaging. If prepared as a dry composition, prior tousage the composition may be prepared as a solution using a suitableliquid, including water and various organic solvents, or mixturesthereof and so on, by techniques well known to those skilled in the art.

Dyes include methylene blue, Tartrazine (CI 19140), Quinoline Yellow (CI47005), Eosin (CI 45380), Acid Phloxine (CI 45410), Erythrosine (CI45430), Sunset Yellow FCF (CI 15985), Acid Violet 5B (CI 42640), PatentBlue AF (CI 42080), Brilliant Cyanine 6B (CI 42660), Acid Brilliant BlueFCF (CI 42090), Naphthalene Green VSC (CI 44025) and Acid Blue Black 10B(CI 20470); and direct dyes such as Paper Yellow GG (CI

Direct Yellow 131), Direct Scarlet 4BS (CI 29160), Congo Red (CI 22120),Violet BB (CI 27905), Direct Sky Blue 5B (CI 24400), Patent Blue Violet,Sulfan Dye), Pentamine, guajazulen blue Pentamine, Phthalocyanine Blue(CI 74180), Black G (CI 35255) and Deep Black XA (CI Direct Black 154).The CI number in the description above indicates the identificationnumber in the Color Index, 3rd Ed., The Society of Dyers and Colorists,Bradford, Yorkshire (1971). Preferred dyes include Isosulfan Blue orother dyes which travel through the lymphatic system.

Chromophores include Fluorescein, Rhodamine, Acid Fuchsin; AcridineOrange; Acridine Red; Acridine Yellow; Alizarin Red; Allophycocyanin;Astrazon Brilliant; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow;Bodipy Fl; Bodipy TMR; Bodipy TR; Calcein; Calcein Blue; Calcium Green;Calcium Orange; Calcofluor White; Cascade Blue; Flazo Orange;Fluorescein Isothiocyanate (FITC); Fura-2; Fura Red; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl YellowSGF; Granular Blue; Lucifer Yellow CH; Lucifer Yellow VS; LysoSensorBlue DND-192, DND-167; LysoSensor Green DND-153, DND-189; LysoTrackerGreen; LysoTracker Yellow; LysoTracker Red; Magdala Red; MagnesiumGreen; Magnesium Orange; Mitotracker Green FM; Mitotracker Orange; NileRed; Nuclear Fast Red; Nuclear Yellow; Oregon Green 488; Oregon Green500; Oregon Green 514; Phorwite AR; Phorwite BKL; Phorwite Rev; PhorwiteRPA; Pontochrome; Blue Black; Procion Yellow; Pyrozal Brilliant;Rhodamine Green; Rhodamine Red; Rhodol Green Fluorophore; Rose Bengal;Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant RedB; Sevron Orange; Sevron Yellow L; Texas Red; Thiozol Orange; True Blue;and Xylene Orange.

In another alternative, particularly suitable for assays in bead format,as described below, phycoerythrin is conjugated to streptavidin, whichbinds tightly and specifically to biotin. Phycoerythrin is a highlyfluorescent protein marker. This alternative is described in FIG. 13.

IV. IMMUNODETECTION METHODS

In certain embodiments, the present invention concerns immunodetectionmethods for binding, purifying, removing, quantifying and/or otherwisegenerally detecting biological components such as a 40-kDa haptoglobinglycoform. Some immunodetection methods include enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometricassay, fluoroimmunoassay, chemiluminescent assay and bioluminescentassay to mention a few. The steps of various useful immunodetectionmethods have been described in the scientific literature, such as, e.g.,Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al.,1993; and Nakamura et al., 1987, each incorporated herein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a 40-kDa haptoglobin glycoform, and contactingthe sample with a first antibody or antibodies that reactimmunologically with the 40-kDa glycoform in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of immunocomplexes.

These methods include methods for purifying a 40-kDa haptoglobinglycoform from tissue samples or other samples from an organism. Inthese instances, the antibody removes the 40-kDa protein (andhaptoglobin) that react immunologically with antibody. The antibody willpreferably be linked to a solid support, such as in the form of a columnmatrix or the well of a plate, and the sample suspected of containingthe 40-kDa haptoglobin glycoform will be applied to the immobilizedantibody. The unwanted components will be washed from the column,leaving the antigen immunocomplexed to the immobilized antibody.

The immunobinding methods also include methods for detecting andquantifying the amount of the 40kDa haptoglobin glycoform in a sampleand the detection and quantification of any immune complexes formedduring the binding process. Here, one would obtain a sample suspected ofcontaining an antigen, and contact the sample with an antibody againstthe antigen, and then detect and quantify the amount of immune complexesformed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing an antigen, such as, for example,a tissue section, or specimen, a homogenized tissue extract, a cell, anorganelle, separated and/or purified forms of any of the aboveantigen-containing compositions, or even any biological fluid that comesinto contact with the cell or tissue, including blood and/or serum,although tissue samples or extracts may be used.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, anyantibodies that react immunologically with anti-tumor antigen antibodiespresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate or dot blot, will generally be washed toremove any non-specifically bound protein species.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological andenzymatic tags appropriate for labeling of a component of theimmunocomplex and detection with a suitable detector. U.S. Patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody and/or a biotin/avidin ligand binding arrangement,as is known in the art.

The antibody or lectin employed in the detection may itself be linked toa detectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. This is a first alternative.Alternatively, the first antibody or lectin that becomes bound withinthe primary immune complexes may be detected by means of a secondbinding ligand that has binding affinity for the antibody. In thesecases, the second binding ligand may be linked to a detectable label.The second binding ligand is itself often an antibody, which may thus betermed a “secondary” antibody. The primary immune complexes arecontacted with the labeled, secondary binding ligand, or antibody, undereffective conditions and for a period of time sufficient to allow theformation of secondary immune complexes. The secondary immune complexesare then generally washed to remove any non-specifically bound labeledsecondary antibodies or ligands, and the remaining label in thesecondary immune complexes is then detected. This is a secondalternative.

Further methods include the detection of primary immune complexes by atwo-step approach. A second binding ligand, such as an antibody, thathas binding affinity for the antibody or lectin is used to formsecondary immune complexes, as described above. After washing, thesecondary immune complexes are contacted with a third binding ligand orantibody that has binding affinity for the second antibody, again undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if this is desired. This is a thirdalternative.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody is used to detect the target antigen(s), and asecond step antibody is then used to detect the biotin attached to thecomplexed biotin. In that method the sample to be tested is firstincubated in a solution containing the first step antibody. If thetarget antigen is present, some of the antibody binds to the antigen toform a biotinylated antibody/antigen complex. The antibody/antigencomplex is then amplified by incubation in successive solutions ofstreptavidin (or avidin), biotinylated DNA, and/or complementarybiotinylated DNA, with each step adding additional biotin sites to theantibody/antigen complex. The amplification steps are repeated until asuitable level of amplification is achieved, at which point the sampleis incubated in a solution containing the second step antibody againstbiotin. This second step antibody is labeled, as for example with anenzyme that can be used to detect the presence of the antibody/antigencomplex by histoenzymology using a chromogen substrate. With suitableamplification, a conjugate can be produced which is macroscopicallyvisible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the Cantor method up to the incubation with biotinylated DNA,however, instead of using multiple rounds of streptavidin andbiotinylated DNA incubation, the DNA/biotin/streptavidin/antibodycomplex is washed out with a low pH or high salt buffer that releasesthe antibody. The resulting wash solution is then used to carry out aPCR reaction with suitable primers with appropriate controls. At leastin theory, the enormous amplification capability and specificity of PCRcan be utilized to detect a single antigen molecule.

The immunodetection methods of the present invention have evidentutility in the diagnosis and prognosis of conditions such as cancerwherein a specific tumor antigen is expressed, and wherein antibodiesexist that react immunologically to an anti-tumor antigen antibody.Here, a biological and/or clinical sample suspected of containing aspecific disease associated antibody is used. However, these embodimentsalso have applications to non-clinical samples, such as in the titeringof antigen or antibody samples, for example in the selection ofhybridomas.

In the clinical diagnosis and/or monitoring of patients with variousforms of a disease, such as, for example, colorectal cancer, thedetection of a cancer specific antigen that reacts and/or an alterationin the levels of such an antigen, in comparison to the levels in acorresponding biological sample from a normal subject, is indicative ofa patient with cancer. However, as is known to those of skill in theart, such a clinical diagnosis would not necessarily be made on thebasis of this method in isolation. Those of skill in the art are veryfamiliar with differentiating between significant differences in typesand/or amounts of biomarkers, which represent a positive identification,and/or low level and/or background changes of biomarkers. Indeed,background expression levels are often used to form a “cutoff” abovewhich increased detection will be scored as significant and/or positive.Of course, the antibodies of the present invention can be used in anyimmunodetection or therapy known to one of ordinary skill in the art.

B. ELISAs

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain immunoassays are the various types ofenzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays(RIA). Other types of immunoassays are known in the art and can be used,including, but not limited to, immunoassays employing lateral flow teststrips, immunoradiometric assays, fluoroimmunoassays, chemiluminescentimmunoassays, bioluminescent immunoassays, enzyme multipliedimmunoassays (EMIT), cloned enzyme donor immunoassays (CEDIA),immuno-PCR assays, phosphor immunoassays, a quantum dot immunoassays,solid phase light-scattering immunoassays, and surface effectimmunoassays. Immunoassays can be competitive or non-competitive and canbe run in a homogeneous or heterogeneous format.

In some aspects of the invention, there are ELISA assays, including inkits, to test samples of subjects that are suspect or at risk for thedevelopment of colorectal cancer. In one exemplary ELISA, theanti-haptoglobin antibodies of the invention are immobilized onto aselected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the antigen, such as a diluted clinical sample, is added tothe wells. After binding and/or washing to remove non-specifically boundimmune complexes, the bound antigen may be detected. Detection can beachieved by contacting the sample with a galactose-binding lectin thatis linked to a detectable label. This type of ELISA is a “sandwichELISA.” Detection may also be achieved by the addition of agalactose-binding lectin, followed by the addition of a further antibodythat has binding affinity for the lectin, with the further antibodybeing linked to a detectable label.

In a further exemplary ELISA, the galactose-binding lectins of theinvention are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing the antigen, such as a dilutedclinical sample, is added to the wells. After binding and/or washing toremove non-specifically bound immune complexes, the bound antigen may bedetected. Detection can be achieved by contacting the sample with ahaptoglobin-binding antibody that is linked to a detectable label.Detection may also be achieved by the addition of a haptoglobin-bindingantibody, followed by the addition of a further antibody that hasbinding affinity for the haptoglobin-binding antibody (e.g., a secondaryantibody), with the further antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either lectin or antibody (e.g., ananti-haptoglobin antibody), one will generally incubate the wells of theplate with a solution of the lectin or antibody, either overnight or fora specified period of hours, such as for 4, 5, 6, 7, 8, 9 or 10 hours.Likewise, the incubation can be performed at a specified temperature,such as between about 1° C. and 22° C., e.g., at 4° C. The wells of theplate will then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein orsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of a lectin or antibody to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with thebiological sample to be tested under conditions effective to allowimmune complex (antigen/antibody) formation. For example, the reactioncould be incubated for 1, 2, 3, 4, or more hours at room temperature.Detection of the immune complex then requires a labeled secondarybinding ligand (e.g., a lectin) or antibody, or a secondary bindingligand or antibody in conjunction with a labeled tertiary antibody or athird binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and/or antibodies with solutions such as BSA, bovine gammaglobulin (BGG) or phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.For example, the inclusion of a detergent such as 0.01 to 0.1% TWEEN-20can significantly reduce non-specific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 20° C. to 27° C., or insome cases overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A particular washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the lectin, second or third antibody willhave an associated label to allow detection. In some cases this will bean enzyme that will generate color development upon incubating with anappropriate chromogenic substrate. In certain aspects the label is anaffinity label such as biotin and detection can be achieved by furthercontact with a detectable affinity molecule (e.g., a labeled avidin).Thus, for example, one will desire to contact or incubate the first andsecond complex with a urease, glucose oxidase, alkaline phosphatase orhydrogen peroxidase-conjugated antibody for a period of time and underconditions that favor the development of further immune complexformation (e.g., incubation for 2 hours at room temperature in aPBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea hydrogen peroxide,or bromocresol purple, or2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H₂O₂,in the case of peroxidase as the enzyme label. Quantification is thenachieved by measuring the degree of color generated, e.g., using avisible spectra spectrophotometer.

As described above, ELISAs are typically performed in a plate format.However, such plate formats are not the only suitable formats forperformance of assays according to the present invention. Otheranalogous assays can be performed in a bead format, such as by the useof a magnetic bead for separation of reagents bound to the bead fromreagents not bound to the bead. An example of a bead format for an assayaccording to the present invention is described in FIG. 13. FIG. 13 is aschematic diagram of the reaction as performed on a bead. Step 1 is thecoupling reaction in which an anti-haptoglobin antibody is coupled to abead. Step 2 is the binding of haptoglobin from serum with and withoutthe galectin-3 ligand to the anti-haptoglobin antibody coupled to thebead. Step 3 is the secondary binding of a biotinylated lectin vector tothe haptoglobin; only the haptoglobin with the galectin-3 ligand bindsto the lectin at this stage. Step 4 is the binding of astreptavidin-phycoerythrin conjugate to the bead; the streptavidin bindsonly to the biotinylated lectin vector bound to the haptoglobin.Phycoerythrin is an intensely fluorescent protein and can be detected byconventional fluorescence detection techniques.

C. Multi-Marker Panels

Another aspect of the present invention is the use of at least oneadditional marker for the detection of colorectal cancer in addition tothe galectin-3-binding haptoglobin glycoform described above. Theseadditional markers include: galectin-3 or, in some alternatives,peptides derived from galectin-3, carcinoembryonic antigen (CEA), andCYFRA21-1. These additional markers, when used in addition to thegalectin-3-binding haptoglobin glycoform, constitute a multi-markerpanel.

Galectin-3 is a protein that in humans is encoded by the LGALS3 gene.

It is a mammalian member of the lectin family The protein is about 30kDa in size and, like all galectins, contains acarbohydrate-recognition-binding domain of about 130 amino acids thatenables the binding of β-galactosides. Overexpression of galectin-3 hasbeen associated with cancer. Changes in subcellular and intercellularlocation of galectin-3 are commonly seen in cancer. The many interactionand binding properties of galectin-3 influence various cellularactivities based on its location, including cancer cell growth anddifferentiation, chemoattraction, apoptosis, immunosuppression,angiogenesis, adhesion, invasiveness, and metastasis. Galectin-3overexpression promotes neoplastic transformation and the maintenance oftransformed phenotypes as well as enhancing adhesion of tumor cells tothe extracellular matrix and increase metastatic spreading. Depending onthe subcellular location of galectin-3, the protein can either inhibitor promote apoptosis. With respect to immune regulation, galectin-3 canregulate immune cell activities and helps contribute to evasion ofimmune surveillance of tumor cells. Galectin-3 may also contribute toangiogenesis of tumors (F.-T. Liu & G. A. Rabinovich, “Galectins asModulators of Tumour Progression,” Nature Rev. Cancer 5: 29-41 (2005)).

In another alternative, peptides derived from trypsin digestion ofgalectin-3 can be used, including IALDFQR (SEQ ID NO: 1), GNDVAFHFNPR(SEQ ID NO:

2), IQVLVEPDHFK (SEQ ID NO: 3), and LDNNWGR (SEQ ID NO: 4), as describedin U.S. Pat. No. 8,945,511 to Weinberger et al.

The detection of galectin-3 or one or more peptides derived fromtrypsin-digestion of galectin-3 can be performed by conventionalimmunological assays known in the art, including, but not limited to, anELISA assay, a radioimmunoassay (RIA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescentimmunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzymedonor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay,a quantum dot immunoassay, a solid phase light-scattering immunoassay, asurface effect immunoassay, and an immunoassay employing lateral flowtest strips Immunoassays can be competitive or non-competitive and canbe run in a homogeneous or heterogeneous format.

Carcinoembryonic antigen (CEA) is actually a group of closely relatedantigens that are glycosyl phosphatidyl inositol (GPI)cell-surface-anchored glycoproteins whose specialized sialofucosylatedglycoforms serve as functional colon carcinoma L-selectin and E-selectinligands. There are at least six proteins that are consideredcarcinoembryonic antigens, known as CD66a, CD66b, CD66c, CD66d, CD66e,and CE66f. Although CEA is expressed by cells of a number of types oftumors, it is particularly associated with adenocarcinomas, includingadenocarcinomas of the colon.

CEA may be also assayed by conventional immunoassays as described above,including the various alternatives described herein.

CYFRA21-1 is also known as cytokeratin 19. This is a 40-kDa proteinencoded by the KRT19 gene. This has been used as a tumor marker.CYFRA21-1 may be assayed by conventional immunoassays as describedabove, including the various alternatives described herein.

In one alternative (Alternative I), a multi-marker panel comprisesgalectin-3 ligand (i.e., the haptoglobin glycoform described above),galectin-3, CYFRA21-1, and CEA. In another alternative (Alternative II),a multi-marker panel comprises galectin-3 ligand, CYFRA21-1, and CEA. Inyet another alternative (Alternative III), a multi-marker panelcomprises galectin-3 ligand and CYFRA21-1. In still another alternative(Alternative IV), a multi-marker panel comprises galectin-3 ligand andCEA. Alternative I is the multi-marker panel with the smallest AIC(Akaike information criterion). Alternative II is the three-marker panelwith the smallest AIC, the three-marker panel with the largest AUC (areaunder the curve), and the best model based on forward selection.

D. Use of Age as a Co-Variate Variable

In yet another alternative, age is used as a co-variate variabletogether with galectin-3 ligand concentration or, if a multi-markerpanel is used, together with the results from the multi-marker panel.

E. Digital Scanning and Uploading of Results

In yet another alternative, the results of quantitation from the finalstep of assay method as described in the present invention are digitallyscanned and uploaded to the server of a medical provider, such as aserver located in a doctor's office, hospital, or clinic, and processedby that server if necessary. The scanning can be performed by adedicated scanner, smartphone, or other digital device with scanningcapability. If the detection of at least one additional marker, asdescribed above, is carried out, the scanning can also include theresults of the detection of the at least one additional marker. Methodsfor such digital scanning and upload are described, e.g., in UnitedStates Patent Application Publication No. 2004/0162690, which isincorporated herein by reference.

F. Detection of Precancerous Conditions

In still another alternative, methods according to the present inventioncan be used to detect or diagnose precancerous conditions of thedigestive tract, such as adenomatous polyps. The adenomatous polyps canbe advanced adenomas or adenomas at an earlier stage, such as smalladenomas. The methods that can be used to detect or diagnose suchprecancerous conditions can either comprise the detection of thegalactose-containing haptoglobin glycoform, as described above, or, thedetection of the at least one additional marker, such as galectin-3, apeptide derived from galectin-3, carcinoembryonic antigen (CEA), andCYFRA21-1, as described above. Age can also be used as a co-variatevariable in such methods as described above.

When methods according to the present invention are used to detect ordiagnose precancerous conditions, the method can further compriseperformance of a polypectomy to treat an adenomatous polyp. Polypectomycan be carried out by conventional surgical methods.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 40-kDA Haptoglobin Glycoform Detection by ELISA

B. Assay Format

Initial results were obtained using a Western blot format to quantify a40-kDa haptoglobin glycoform and galectin-3 ligand in sera, however thisformat was not suitable for a diagnostic assay. For example,disadvantages of initial assay include that it was very time-consumingand did not yield a linear response that could be used to quantify the40kDa haptoglobin glycoform in a sample, i.e., 40-kDa band density wasnot directly proportional to amount of the glycoform. Likewise theWestern blot assay exhibited gel-to-gel variability and required a knownpositive cancer serum as a common reference serum, which is notpractical for scale-up.

A sandwich assay was developed to allow measurement of the 40-kDaglycoform in a complex background by immobilizing ligand with a“catcher” antibody or lectin and detecting the immobilized ligand with a“tracer” antibody or ligand. Two complementary sandwich ELISA formatsusing anti-haptoglobin and lectin were extensively evaluated. A sandwichenzyme-linked immunosorbent assay using lectin catcher andanti-haptoglobin tracer was employed in early developmental studies.However, the alternative sandwich ELISA, using anti-haptoglobin catcherand lectin tracer was found to be superior in discriminating knowncancer sera from normal specimens, and was therefore selected forfurther development and clinical validation (FIG. 2).

C. Lectin Selection

Initial attempts were made to develop a lectin/anti-haptoglobin sandwichELISA assay using biotinyl-galectin-3. Although biotinyl-galectin-3 waseffective for detection of the 40-kDa glycoform on Western blots, it wasnot sufficiently sensitive to use as a reagent for the sandwich ELISA.It was found that the failure of biotinyl-galectin-3 to bind toasialohaptoglobin in a microplate format reflected a loss ofcarbohydrate binding, and was not due to gross degradation of galectin-3or to inability of secondary reagents to detect biotinyl-galectin-3.

Preliminary studies indicated that Erythrina cristagalli lectin (ECL)had a specificity similar to the mammalian lectin galectin-3. Therefore,ECL was used to develop a sandwich ELISA. Both the ECL/anti-haptoglobinand anti-haptoglobin/biotinyl-ECL formats detected asialo-haptoglobin inserum samples, but the anti-haptoglobin/biotinyl-ECL sandwich ELISA wasmore specific for known cancer specimens. Otherbeta-galactoside-specific plant lectins that were tested included thosefrom Ricinus communis, Sophora japonica, Datura stramonium, andLycopersicon esculentum. However, none of these appeared superior to ECL(Table 1).

TABLE 1 Sensitivity and Specificity of Beta-Galactoside Specific Lectinsin Sandwich ELISAs Lectin Sensitivity Specificity Accuracy Erythrinacristagalli 70% 50% 60% Ricinus communis 40% 40% 40% Datura stramonium60% 60% 60% Lycopersicon esculentum 40% 60% 50%

One key step necessary for the successful development of the sandwichELISA is the method used for desialylation. Various studies wereundertaken to address the necessity for desialylation, the relativeefficacy of neuraminidase enzyme vs. mild acid, and the effect ofdilution prior to mild acid treatment.

D. Desialylation

Initial studies established that desialylation was necessary for bindingof galectin-3 to the 40 kDa glycoform, but a desialylation procedureneeded to be developed that could yield quantitative assay results. Indeveloping the sandwich ELISA, it was found that desialylation wasnecessary for binding of ECL to haptoglobin. Initially, desialylationwas performed either with Vibrio cholerae neuraminidase digestion orwith mild acid hydrolysis (0.1 N H₂SO₄, 60 min at 80° C.) with minimaldilution (1.25-fold). Sandwich ELISAs, using either ECL/anti-haptoglobinor anti-haptoglobin/ECL indicated that the neuraminidase digestion gaveat least a 10-fold higher signal than the mild acid hydrolysis treatment(FIG. 1). Therefore, early assays of known cancer and normal sera wereperformed using neuraminidase digestion, but there was difficulty ingetting reproducible results.

To further develop the desialylation method, the loss of antigenicity ofhaptoglobin caused by mild acid treatment was studied in detail. Directbinding assays established that this was due to loss of binding ofanti-haptoglobin to the mild-acid-treated glycoprotein. Some of thisloss of antigenicity could be due to concentration-dependentaggregation, thus, the mild acid treatment was repeated at higherdilutions (5-fold, 25-fold, and finally 100-fold dilution). With themild acid hydrolysis performed at higher dilutions, signal wasequivalent to that obtained with neuraminidase digestion, with a higherspecificity for cancer sera (FIG. 2). Desialylation by mild acidhydrolysis at a 100-fold dilution was therefore used in clinical methodvalidation.

E. Additional Element of the ELISA

Two different anti-haptoglobin antibodies were evaluated for potentialuse in sandwich ELISA, a polyclonal anti-haptoglobin and a mousemonoclonal anti-haptoglobin. The two antibodies gave similar results inthe ECL/anti-haptoglobin sandwich ELISA format, but only the polyclonalanti-haptoglobin could practically be used in theanti-haptoglobin/biotinyl-ECL format.

Concentrations of anti-haptoglobin used as catcher, or biotinyl-ECL usedas tracer, and of ABC complex (avidin-biotin complex) were varied toensure they were not limiting. In addition, the effects of blocking witha commercial block solution rather than with bovine serum albumin (BSA)and of diluting sera in PBS rather than PBST, were studied, however, BSAand PSBT were found to be superior. Extensive studies with known cancersera and known normal sera were also performed to determine the optimal20,000-fold dilution of desialylated serum that would fall within thelinear range of the assay.

Example 2 Detailed 40-kDa Haptoglobin Glycoform Detection Protocol

F. Mild Acid Hydrolysis and Dilution:

For initial sample preparation the following steps were used: Serum orplasma specimens were thawed (aliquotted in small volumes (0.3 ml), thenstored at −80° C. 5-μL samples of coded serum were collected. Residualaliquots were marked as once-thawed, and return excess to −80° C.Samples were added to 395 μL water in 1.5-mL tube and mixed. 100 μL ofH₂SO₄ was added and mixed with the diluted samples. Samples were heatedfor 60 min at 80° C. and then cooled on ice. 100 μL of 10× PBS was addedand mixed with the samples. 100 μL of 0.5 N NaOH was added and mixedwith the samples. 300 μL of water was added and mixed with the samples.The pH of the samples were checked (with pH paper) and recorded.Resulting 200-fold diluted desialylated serum samples were refrozen in200-μL aliquots at −20° C. Three aliquots are used on separate days forELISA. Two aliquots are reserved.

G. Preparation of Anti-Haptoglobin Coated Plates

Day 1

Four 96-well microtiter plates were marked with triplicate wells insidefor serum specimens, PBST blanks and normal asialohaptoglobin standards.Typical plate geometry is shown in Table 2.

100 μL Rabbit anti-haptoglobin (Sigma Catalog #H-8636) was diluted into25 mL PBS and 50 μL was added to each well of four microtiter plates.The plate is left overnight at 4° C.

Day 2

Anti-haptoglobin is decanted and discarded. Excess liquid is removed bystriking the inverted plate on paper towels. Each well is then washedonce with PBS using a squeeze bottle. The wash is decanted anddiscarded. Again, excess liquid is removed by striking the invertedplate on paper towels. 200 μL of 1% BSA in PBS (prepared fresh daily)was added to each well and the components left for 60 minutes at roomtemperature. Meanwhile, final dilutions of standards, unknowns, andcontrols were prepared. BSA/PBS was decanted and discarded. Excessliquid is removed by striking the inverted plate on paper towels.

Final Dilutions of Standards, Unknowns, and Controls

Final dilutions of coded desialylated serum were produced by thawingfresh aliquots of 56 coded specimens of 200-fold diluted desialylatedserum. 5 μL taken and the residual aliquot was marked as once-thawed,and returned to −20° C. The 5 μL sample was added and mixed to 495 μL ofPBST on ice in a 500-μL tube to give 20,000-fold final dilution relativeto serum.

Final dilutions of known positive and negative specimens were producedby thawing re-frozen aliquots of 2 known positive cancer specimens(200-fold diluted desialylated serum) or re-frozen aliquots of 2 knownnegative normal specimens (200-fold diluted desialylated serum),respectively. 10-μL samples were removed from each specimen and theresidual aliquots returned to −20° C. The 10 μL samples were each addedto and mixed with 990 μL PBST on ice in 1.5 mL tubes to give 20,000-foldfinal dilution relative to serum.

Final dilution of normal asialohaptoglobin standards were produced bythawing 50 μg/ml stock of normal asialohaptoglobin (asHP). Aftersampling, the residual stock was returned to −20° C. A 20-μL aliquot of50 μg/mL asHP was added and mixed with 1980 μL PBST in a 15-mL tube togive 500 ng/mL asHP. A 400 μL sample of 500 ng/mL asHP was added andmixed with 600 μL PBST in a 1.5 mL tube to give 200 ng/mL asHP. A 200-μLsample of 500 ng/ml asHP was added to and mixed with 800 μL PBST in a1.5 mL tube to give 100 ng/mL asHP. A 100 μL sample of 500 ng/mL asHPwas added to and mixed with 900 μL PBST in a 1.5 mL tube to give 50ng/mL asHP. A 40 μL sample of 500 ng/mL asHP was added to and mixed with960 μL PBST in a 1.5 mL tube to give 20 ng/mL asHP. Finally, a 20 μLsample of 500 ng/mL asHP was added to and mixed with 980 μL PBST in 1.5ml tube to give 10 ng/mL asHP.

Binding of Analytes and Standards

50 μL/well of 20,000-fold diluted unknown desialylated serum in PBST wasadded in triplicate (3×16=48 interior wells/plate). 50 μL/well of20,000-fold diluted known control sera in PBST was added in triplicate(3×4=12 wells/plate). 50 μL/well of PBST was added to 24 wells (6interior and 18 exterior) as blanks. 50 μL/well of asHP standards (10ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, & 500 ng/mL) was added,in triplicate (3×6=18 exterior wells/plate). Again, typical plategeometry is shown in Table 2. The plate was then incubated for 1 hour atroom temperature. Meanwhile, 1 μg/mL biotinyl Erythrina cristagallilectin was prepared in PB ST. Excess liquid was removed and discardedfrom the plate by striking the inverted plate on paper towels. Each wellwas washed once with PBST (using a squeeze bottle), and excess liquidwas removed and discarded by striking the inverted plate on papertowels.

Biotinyl Lectin Binding

50 μL/well of 1 μg/mL biotinyl Erythrina cristagalli lectin in PBST wasadded. The plate was incubated for 1 hour at room temperature.(Meanwhile, the Avidin-Biotin-Complex was prepared.) Excess liquid isremoved and discarded by striking the inverted plate on paper towels.Each well is washed with PBST a first time (using a squeeze bottle).Excess liquid from the first wash is removed by striking the invertedplate on paper towels. Each well is washed with PBST a second time(using a squeeze bottle). Excess liquid from the second wash is removedby striking the inverted plate on paper towels. Each well is washed withPBST a third time (using a squeeze bottle). Excess liquid from the thirdwash is removed by striking the inverted plate on paper towels.

Preparation of Avidin-Biotin Complex

A VECTASTAIN ABC Reagent Kit (Vector Elite PK-6100) was used fordetection. 10 drops of REAGENT A (Avidin DH) was added to 25 mL PBST,then 10 drops of Reagent B (biotinylated horseradish peroxidase) wasadded. Reagents were mixed and allowed to stand 30 min at roomtemperature.

Detection of Biotinyl Lectin Bound

50 μL/well of Avidin-Biotin Complex was added. The plate was left for 1hour at room temperature, during which the ABTS reagent was prepared.Excess liquid was removed and discarded by striking the inverted plateon paper towels. Each well was then washed with PBST three times (usinga squeeze bottle). Excess liquid from each wash was removed by strikingthe inverted plate on paper towels. 100 μL/well of ABTS reagent (0.03%H₂O₂, 1 mM 2,2-azino-di(3-ethylbenzthiazoline) sulfonate in 0.1 Mcitrate, pH 4.0) was added and the time noted. The plate was incubatedfor exactly 30 minutes at room temperature. The plate was then read atA₄₀₅ on a Dynex Technologies MRXTC ELISA reader.

Calculations

For each plate, the median blank was subtracted from gross A₄₀₅ ofstandards, controls, and unknowns. A regression line was constructed foreach plate with the 1 ng to 10 ng standards (ignoring 0.5 ng and 25 ngstandards) to use for mg/ml calculations. For each plate, mg/mLconcentrations are calculated for each unknown and control median netA₄₀₅ values from triplicate wells using the regression line. Median netA₄₀₅ values from triplicate wells were used to calculate mg/mL for eachunknown. Any excessive plate-to-plate variation in the blank, standards,or known controls was noted.

Each serum was assayed on 3 separate plates on separate days (in batchesof 4 plates). For each serum, the mg/mL results of first assay(sequential order of samples), second assay (shuffled order), and thirdassay (scrambled order) were presented individually, along with medianand the mean, SD, and SEM of the mg/mL results. Any excessive day-to-dayvariation in blank, standards, or known controls was noted.

Reagents and Reagent Preparation for Assays

Rabbit anti-haptoglobin antibody was purchased from Sigma (catalog#H-8636). Bovine serum albumin (BSA) was purchased from Sigma (catalog#A-3059). Biotinyl ECL was purchased from Vector Laboratories (catalog#B-1145). VECTASTAIN® Elite® Avidin Biotin Complex (ABC kit) waspurchased from Vector Laboratories (catalog #PK-6100). Phosphatebuffered saline (PBS) was prepared in-house from a 10× PBS solution of137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, and 1.5 mM KH₂PO₄. PBST wasprepared by adding 0.05% TWEEN-20 (Sigma Catalog #P3563) to PBS.

The Asialohaptoglobin standard was prepared by adding 500 μL of 1 mg/mLhaptoglobin (Sigma catalog #H-3536) in water to 300 μL water in a 15-mLtube and mixing. 200 μL of 0.5 N H₂SO₄ was added and mixed. The mixturewas then heated for 60 minutes at 80° C. and cooled on ice. 1 mL of 10×PBS was added and mixed. 200 μl of 0.5 N NaOH was added and mixed. 8.8ml water was added, mixed and the pH of the resulting solution checkedfor neutralization with pH paper. The resulting 50 μg/mlasialohaptoglobin standard was frozen in 200 μL aliquots at −20° C.

The ABTS reagent (0.03% H₂O₂, 1 mM 2,2-azino-di(3-ethylbenzthiazoline)sulfonate in 0.1 M citrate (pH 4.0)) was prepared by mixing 30% hydrogenperoxide (Sigma catalog #H-1009) (diluted 1000-fold for ABTS reagent,100 mM ABTS in water (Sigma catalog #A-1888) (store frozen in 500 μLaliquots; diluted 100-fold for ABTS reagent), and 1 M citrate pH 4.0(dilute 10-fold for ABTS reagent).

Example 3 Assessment of Assay Sensitivity, Specificity, andReproducibility

The high throughput ELISA assay detailed above takes advantage ofsimilarities in ligand binding between galectin-3 and the lectinErythrina cristagalli. This sandwich ELISA was used to compare 150blinded sera samples from the Early Detection Research Network (EDRN)colon reference set (normal controls, adenomas and adenocarcinomas).Results of the assays were used to constructed receiver operatingcharacteristic curves of sensitivity versus (1-specificity). The curvesshown in FIG. 3 demonstrated that the assay successfully differentiatedindividuals with colorectal neoplasia from normal controls with a highdegree of sensitivity and specificity.

The AUC for the galectin-3 (Ga13) ligand alone, normal versus cancer,was 0.84 and for Gal3 ligand+FOBT (fecal occult blood test) was 0.91(FOBT alone 0.62). The AUC for Gal3 ligand alone, normal versus allneoplasia (adenoma+carcinoma), was 0.74 and for Gal3 ligand plus FOBTnormal versus all neoplasia was 0.80. Thus, based on a newly developedassay, the serum 40-kDa haptoglobin glycoform (and galectin-3 ligand)shows promise for validation as a clinically relevant biomarker fordetection of colorectal neoplasia. The ELISA was validated within andbetween days using calibration curves and concentration reproducibility.Each unknown serum was assayed on 3 separate plates on 3 separate days.

These data demonstrate that the assay is linear to 500 pg. Analyticsamples assayed on three different days were within a coefficient ofvariability of 15% (see, e.g., FIG. 4). Day to day variation in assayresults was assessed in more detail in the studies shown in FIG. 5. Asshown, while absolute quantitation of serum 40-kDa haptoglobin glycoformvaried slightly from day to day, discrimination between cancer andcontrol samples was maintained in all cases.

It was further demonstrated that assay results remained consistent overtime and when samples were stored (frozen) for extended periods of timeprior to being subjected to assay. As shown in FIG. 6, samples werequantitatively assessed for the 40-kDa serum haptoglobin glycoform andthen stored for three years prior to reassessment. Despite the storagetime and the time elapsed between assays the 40-kDa glycoform levelsfrom the original assay and the reassessment correlated well. Thesestudies demonstrate not only that accurate results can be obtained fromstored frozen samples, but also that the assay system was highlyreproducible.

Example 4 High-Throughput Assay Modification

In order to adapt the assay for use in a high throughput format, theELISA based system was modified for use with beads. For one couplingreaction, 100 μL of uncoupled magnetic beads (MC10026-01 Bio-plex ProMagnetic COOH Beads, Region 26, 1 mL, BioRad) at the concentration of1.25×10⁷ beads/mL were put into a tube, after vortex and sonication(mini UltraAsonik™ Ney) steps. The tube was positioned in a magneticseparator containing a strong magnet for 1 minute. Then, the supernatanthad to be delicately removed and 100 μL of wash buffer (PBS, 0.05%TWEEN-20, pH 7.4) was added to the uncoupled beads. The mixture waspositioned in the magnetic separator for 1 minute and the supernatantwas discarded. The uncoupled beads were then resuspended in 80 μL ofactivation buffer (0.1 M NaH₂PO₄, pH 6.2). 10 μL of fresh 50 mg/mL S-NHS(Thermo Scientific, Prod. #24510) in activation buffer are added to thetube followed by the fresh addition of 10 μL of 50 mg/mL EDC (ThermoScientific, Prod #22980). The beads were activated for 20 min at RTunder agitation and in the dark. After that, activated beads were washedtwice with 150 μL of PBS with a high speed mix for 10 seconds after eachwash. Between each wash, the supernatant was removed after 1 minute inthe magnetic separator. The activated beads were resuspended with 100 μLof PBS, and 9 μg of anti-haptoglobin antibody diluted in PBS was addedto the tube. The total volume was brought to 500 μL with PBS and theactivated beads were incubated for 2 hours at room temperature underagitation and in the dark. After the 2 hours of coupling, the tube waspositioned in the magnetic separator for 1 minute. The supernatant wasthen discarded and the coupled beads were resuspended with 500 μL ofPBS. The coupled beads were incubated 30 minutes at room temperature,under agitation and in the dark, with 250 μL of blocking buffer (PBS, 1%BSA, 0.05% azide, pH 7.4). Finally, the tube was positioned in themagnetic separator for 1 minute and the supernatant was removed. Thecoupled beads were then resuspended in 650 μL of storage buffer (PBS,0.1% BSA, 0.02% TWEEN-20, 0.05% azide, pH 7.4) and the beadconcentration was estimated with a hemocytometer.

To a 96-well plate (Greiner Bio-One, No. 655096), anti-haptoglobincoupled beads (2500 beads/well) were added. Then, 50 μL of haptoglobinstandards or samples were added to each well. The wells were homogenized(Mixer type 16700) and the plate was incubated 1 hour at roomtemperature under agitation and in the dark. After that, the plate waswashed twice with PBST (Bio-Plex Pro™ Wash Station). Then, 50 μL ofbiotinyl-ECL at the concentration of 2.5 mg/mL prepared in stainingbuffer (PBS, 1%; BSA, pH 7.4) was added to each well. The plate wasmixed and was incubated for 1 hour at room temperature under agitationin the dark. The plate was then washed three times with PBST andfinally, 5 μL of streptavidin-PE (phycoerythrin) (BioRad #171-304501) atthe concentration of 2 μg/mL prepared in staining buffer were added toeach well. The plate was mixed and incubated for 10 minutes at roomtemperature under agitation and in the dark. Finally, 75 μL of storagebuffer was added to each well and after 2 minutes of plate agitation,the streptavidin-PE fluorescence on beads was read by a Bio-plexinstrument (BioRad, CA).

As shown in FIG. 7, assays performed on beads resulted in similarspecificity and sensitivity as compared to assays completed in wells ofmicrotiter plates (see, e.g., FIG. 3 vs. FIG. 7). Thus, these studiesdemonstrate that the assays can be replicated on the surface of beadsand are thus amenable to further commercial scale-up.

FIG. 13 shows a schematic diagram of the reaction as performed on abead. Step 1 is the coupling reaction in which an anti-haptoglobinantibody is coupled to a bead. Step 2 is the binding of haptoglobin fromserum with and without the galectin-3 ligand to the anti-haptoglobinantibody coupled to the bead. Step 3 is the secondary binding of abiotinylated lectin vector to the haptoglobin; only the haptoglobin withthe galectin-3 ligand binds to the lectin at this stage. Step 4 is thebinding of a streptavidin-phycoerythrin conjugate to the bead; thestreptavidin binds only to the biotinylated lectin vector bound to thehaptoglobin.

Example 5 Use of Multi-Marker Panel for Detection of Colorectal Cancerand Adenomatous Polyps

Seven blood-based markers were evaluated. These markers included: (1)galectin-3 ligand (the haptoglobin glycoform described above); (2)galectin-3; (3) carcinoembryonic antigen (CEA); (4) CYFRA21-1; (5)MAPRE16; (6) ferritin; and (7) CRP16 using 2 independent well annotatedsample sets (Early Detection Research Network GLNE sample set and DanishEndoscopy Trial II set). The EDRN sample set included samples from 300individuals including samples from 100 normals, 100 cancers (50 earlyand 50 late stage), and 100 adenomas (50 advanced, 50 non-advanced). TheDanish Endosocopy II trial included a similar set of 450 samples binnedby diagnosis and annotated for potential confounding factors such asage, metabolic syndrome, diabetes, and smoking as well as othernon-cancer findings.

When galectin-3 ligand was used alone, galectin-3 ligand demonstratedperformance in differentiating normal individuals from those with cancer(all stages) and advanced adenomas with blinded verification acrosssample sets (AUCs vs normal for the EDRN sample set include 0.80 allcancer, 0.84 stages III +IV cancer, 0.77 stages I +II cancer, and 0.78advanced adenomas). While impressive, this single marker data, however,suggested that additional sensitivity at high specificity would berequired for a clinically relevant blood test. Combinations ofadditional markers were therefore studied to successfully developmulti-marker blood test panels with higher sensitivity at highspecificity (85% to 95% specificity) required for a clinically relevanttest using the EDRN sample set (and verified with Endoscopy II samples).Combinations of markers similar to that studied using the EDRN sampleset demonstrated superior performance for differentiating not onlycancer from normal individuals, but individuals with colorectal cancerfrom a wide variety of individuals without cancer, including those withpotential confounding factors such as metabolic syndrome, diabetes, andsmoking as well as other non-cancer findings. Furthermore, using samplesfrom Endoscopy II it was demonstrated that age added as a co-variate togalectin-3 ligand resulted in a test superior to galectin-3 ligandalone.

FIG. 8 shows ROC curves for galectin-3 ligand alone for differentiatingnormal individuals without cancer from other groups: (A): advancedadenoma; (B) early stage cancer (stages I and II); (C) late stage cancer(stages III and IV); and (D) all cancers.

FIG. 9 shows performance of marker panels by patient group showing AUCwith sensitivities at 85% and 90% specificities; the patient groupsversus normal are: (i) all colorectal cancer (CRC); (ii) advancedadenoma; (iii) stage I and II CRC; (iv) stage I, II, IIIA, and IIIB CRC;(iv) stage IIIC and IV CRC; and (v) SRN (all CRC and advanced adenoma).The models shown in FIG. 9 are: (i) galectin-3 ligand (Model 0); (ii)galectin-3 ligand, CEA, CYFRA21-1, and galectin-3 (Model 1); (iii)galectin-3 ligand, CEA, and CYFRA21-1 (Model 2); (iv) galectin-3 ligandand CYFRA21-1 (Model 3); and (v) galectin-3 ligand and CEA (Model 4).The data for advanced adenomas using multi-marker panels found in FIG. 9can be compared with optimal data in the literature for commonly usedstool-based tests which suggests 23.8% sensitivity (AUC 0.67) for fecalimmunochemical testing (FIT) and 42.4% (AUC 0.73) for Cologuard® fordetecting “advanced precancerous lesions” (NEJM 2014; 370:1287), and 11%sensitivity for the methylated septin 9 (PRESEPT®) blood-based test.

FIG. 10 shows ROC curves for: Models 1, 2, 3, and 4: for all cancers vs.normal (Panel A); advanced adenoma vs. normal (Panel B); stage I and IIof colorectal cancer vs. normal (Panel C); stage I, II, and III ofcolorectal cancer vs. normal (Panel D); stage IV of colorectal cancervs. normal (Panel E); and all colorectal cancers and advanced adenoma(Panel F).

Data from the Danish Endoscopy II trial specimens was used as aconfirmation of the data generated using the EDRN specimens. It differsfrom the EDRN data in that it included as controls patients of variousages as well as “normal” individuals with co-morbidities such asmetabolic syndrome, diabetes, and findings such as diverticulitis orother relevant findings (but no cancer). These co-morbidities have beenfound in many cases to reduce the performance of markers (includingblood-based markers) in differentiating patients with colorectal cancerfrom other groups. Despite this using a multi-marker panel similar toModel 2 above (galectin-3 ligand, CYFRA21-1, CEA), this multi-markerpanel demonstrated high performance in differentiating those withcolorectal cancer from all other groups (AUC 0.82). FIG. 11 shows ROCcurves for Model 2 for all cancers vs. normal using data from DanishEndoscopy II trial specimens.

Use of the Endoscopy II samples also demonstrated the superiority ofusing age as a co-variate (age+galectin-3 ligand versus galectin-3ligand alone). In FIG. 12 solid curves represent galectin-3 ligand alonewhile dashed curves represent galectin-3 ligand+age as a co-variate.Panel A is small adenomas vs. normals without comorbidity. Panel B isadvanced adenomas vs. normals without comorbidity. Panel C is alladenomas vs. normals without comorbidity. Panel D is early cancer vs.normals without comorbidity. Panel E is late cancer vs. normals withoutcomorbidity. Panel F is all cancer vs. normals without comorbidity.

FIG. 14 is a table showing the performance sensitivity and specificityof a bead assay for galectin-3 ligand, with breakdown of the subjects asnormal, small adenoma, advanced adenoma, cancer at Stage I/II, cancer atStage IIIA/B, or cancer at Stage IIIC/IV. In the results shown in FIG.14, the performance of a bead-based ELISA for galectin-3 ligand wasevaluated in serum samples from 295 patients individuals obtained fromNCI's Early Detection Research Network. This sample set included serafrom subjects with normal colonoscopies (n=94), small non-advancedadenomas (n=50), advanced adenomas (n=51), colorectal cancer (CRC)stages I/II (n=50), CRC stages IIIA/IIIB (n=27), and CRC stages IIIC/IV(n=23). FIG. 15 is a graphical representation of the data from FIG. 14.

FIG. 16 shows the performance of plate and bead assays for galectin-3ligand for high-risk adenomas, cancer at Stage I/II, cancer at StageIII/IV, or all cancers. The performance of plate and bead-based ELISAsfor galectin-3 ligand was further validated in an independent set ofserum samples from The Danish Endoscopy Study II trial. Serum sampleswere collected prior to first-time colonoscopy from individuals withcolon cancer, adenomas and normal controls. When 50 specimens frompatients diagnosed as Normal (no comorbidity) are compared to specimensfrom patients diagnosed as adenoma—high risk (>=10mm, n=78), stage I andII CRC (n=33) and stage III and IV CRC (n=30), ROC analysis of both thebead assay and the plate assay broadly confirmed the sensitivity andspecificity seen for the EDRN GLNE data set, as shown in the table ofFIG. 16. The performance of the plate and bead assays were essentiallyequivalent.

The data presented in FIG. 17 show the performance of individual cancermarkers by patient group (advanced adenoma, early colorectal cancer,late colorectal cancer, all colorectal cancer, and colorectal cancerplus advanced adenoma). The individual cancer markers analyzed aregalectin-3 ligand (i.e., the haptoglobin glycoform), carcinoembryonicantigen (CEA), galectin-3, MAPRE-1, ferritin, CYFRA21-1, and CRP16. Itis clear from the results of FIG. 17 that, considered individually, themost efficient single marker is the galectin-3 ligand.

FIG. 18 is a table showing a comparison of the results with thegalectin-3 ligand test in the bead format with and without age as acovariate for small adenomas, advanced adenomas, all adenomas, cancerStage I/II, cancer Stage III/IV, and all cancers. Consideration of ageas a covariate improves the efficiency of the assay in the bead formatfor all stages considered.

A confirmatory study was performed by examining sample sets obtainedfrom various sources to demonstrate that the galectin-3 ligand assay candifferentiate cancer or pre-cancer from normal. Over 5,000 patientsamples were run from a prospective colon cancer screening trial (GLNE10) for galectin-3 ligand. In addition, galectin-3 ligand was assayed in500 patient samples from the prospective Danish Endoscopy 3 colon cancerscreening trial. This sample set contains serum from normal patients,patients with pre-cancerous polyps and patients with colon cancer. Thedistribution of galectin-3 ligand measurements is shown in FIG. 20.These samples were also run for all of the other markers describedherein.

In addition, another methodologic platform (lateral flow) was performedto measure galectin-3 ligand and CEA in pooled colon cancer and normalsera and the results confirmed the ability of the galectin-3 ligandmarker to differentiate cancer from normal.

It was found that blinded analysis of an additional 191 cancers and 291normal from the independent Mayo set yielded a galectin-3 ligand singlemarker AUCs similar to that derived from previous sample sets (AUC earlystage cancer versus normal 0.743). Further, blinded analysis of anadditional sample set consisting of 133 cancers and 175 normals obtainedfrom Flinders Hospital in Australia yielded a galectin-3 ligand singlemarker AUC's (cancer versus normal 0.75 with significant differencesversus normal for all stages of cancer). This study also confirmed theability of this assay to measure this marker in serum or plasma.

In addition, pooled sera from 35 patients with colorectal cancers and 35with normal colonoscopies were assayed blindly using lateral flowtechnology. The successful detection of galectin-3 ligand from serum, ina point-of-care, lateral flow assay format was demonstrated. Acidtreated samples were interrogated by LFA and compared to a standardcurve as shown in FIG. 19. The calculated ligand concentrations wereobtained; Cancer—7.94 mg/ml, Normal 1.17 mg/ml. The resulting DC/DNratio was 6.79, higher than obtained by ELISA. 500 blinded samplesobtained from Denmark's Endoscopy 3 prospective colon cancer screeningtrial were assayed for galectin-3 ligand. While the data remains blindedthe distribution of measurements for galectin-3 ligand is in keepingwith differing levels among patient based on findings (FIG. 20).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for detection and/or diagnosis ofcolorectal cancer in a subject comprising: (a) obtaining a serum samplefrom the subject; (b) desialylating the serum sample; (c) contacting thedesialylated serum sample with an antibody that binds haptoglobin toform a complex; and (d) quantitating the complex with a detectablelectin that binds galactose in order to detect or diagnose colorectalcancer using a quantitative immune-detection assay, wherein the presenceof a galactose-containing haptoglobin glycoform is associated with thepresence of colorectal cancer, wherein the quantitative immune-detectionassay is not a Western blot.
 2. The method of claim 1, wherein the serumis diluted between about 25-fold to about 50-fold prior todesialylation.
 3. The method of claim 1, wherein the serum is diluted atleast 50-fold prior to desialylation.
 4. The method of claim 1, whereinthe desialylated serum is further diluted to a final dilution of betweenabout 100-fold and 150,000-fold; of between about 1,000-fold and about150,000-fold, or about 15,000-fold and about 150,000-fold before step(c).
 5. The method of claim 4, wherein the desialylated serum is furtherdiluted to a final dilution of about 20,000-fold before step (c).
 6. Themethod of claim 1, wherein the quantitative immune-detection assay is anELISA assay, a radioimmunoassay (RIA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescentimmunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzymedonor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay,a quantum dot immunoassay, a solid phase light-scattering immunoassay, asurface effect immunoassay, or an immunoassay employing lateral flowtest strips.
 7. The method of claim 6, wherein the quantitativeimmune-detection assay is an ELISA.
 8. The method of claim 7, whereinthe ELISA is a sandwich ELISA.
 9. The method of claim 1, whereindesialylating the serum comprises treating the serum with a mild acid.10. The method of claim 9, wherein the mild acid is H₂SO₄.
 11. Themethod of claim 1, wherein desialylating the serum comprises treatingthe serum with a neuraminidase.
 12. The method of claim 1, wherein theantibody that binds haptoglobin is a polyclonal antibody.
 13. The methodof claim 1, wherein the antibody that binds haptoglobin is bound to asubstrate or a magnetic bead.
 14. The method of claim 12, wherein theantibody that binds haptoglobin is a polyclonal antibody raised againstpurified human haptoglobin.
 15. The method of claim 1, wherein themethod further comprises a step of contacting the complex of step (c)with a wash solution prior to step (d).
 16. The method of claim 15,wherein the wash solution comprises a detergent.
 17. The method of claim1, wherein the detectable lectin that binds galactose is selected fromthe group consisting of Ricinus communis lectin, Sophora japonicalectin, Datura stramonium lectin, Erythrina cristagalli lectin, andLycopersicon esculentum lectin.
 18. The method of claim 17 wherein thedetectable lectin is Erythrina cristagalli lectin.
 19. The method ofclaim 1, wherein quantitating the complex with a detectable lectincomprises detecting an enzymatic activity.
 20. The method of claim 19,wherein the lectin is biotinylated or comprises a conjugated enzyme. 21.The method of claim 20, wherein the lectin is biotinylated and detectingthe complex comprises contacting the complex with an avidin-reporterconjugate.
 22. The method of claim 21, wherein the reporter is anenzyme.
 23. The method of claim 22, wherein the enzyme is selected fromthe group consisting of urease, alkaline phosphatase, horseradishperoxidase, glucose 6-phosphate dehydrogenase, β-galactosidase, andglucose oxidase.
 24. The method of claim 1 further comprising the stepof using age as a co-variate variable to increase the sensitivity andspecificity of the method.
 25. The method of claim 1 wherein thequantitation of step (d) is digitally scanned and uploaded to the serverof a medical provider.
 26. The method of claim 1 wherein thequantitative immune-detection assay is performed using a magnetic bead.27. The method of claim 26 wherein the quantitative immune-detectionassay is performed using detection of phycoerythrin conjugated tostreptavidin.
 28. A method for detection and/or diagnosis of colorectalcancer in a subject comprising: (a) obtaining a serum sample from thesubject; (b) desialylating the serum sample; (c) contacting thedesialylated serum sample with an antibody that binds haptoglobin toform a complex; (d) quantitating the complex with a detectable lectinthat binds galactose in order to detect or diagnose colorectal cancerusing a quantitative immune-detection assay; and (e) assaying the samplefor the quantity of at least one additional marker to confirm thedetection or diagnosis of colorectal cancer using one or more additionalquantitative immune-detection assays.
 29. The method of claim 28 whereinthe at least one additional marker is at least one of galectin-3, apeptide derived from galectin-3, carcinoembryonic antigen (CEA), andCYFRA21-1.
 30. The method of claim 29 wherein the at least oneadditional marker is galectin-3, CYFRA21-1, and CEA.
 31. The method ofclaim 29 wherein the at least one additional marker is CYFRA21-1 andCEA.
 32. The method of claim 29 wherein the at least one additionalmarker is CYFRA21-1.
 33. The method of claim 29 wherein the at least oneadditional marker is CEA.
 34. The method of claim 29 wherein the one ormore additional quantitative immune-detection assays for assay of thequantity of the at least one additional marker are selected from thegroup consisting of an ELISA assay, a radioimmunoassay (RIA), animmunoradiometric assay, a fluoroimmunoassay, a chemiluminescentimmunoassay, a bioluminescent immunoassay, an enzyme multipliedimmunoassay (EMIT), a cloned enzyme donor immunoassay (CEDIA), animmuno-PCR assay, a phosphor immunoassay, a quantum dot immunoassay, asolid phase light-scattering immunoassay, a surface effect immunoassay,and an immunoassay employing lateral flow test strips.
 35. The method ofclaim 34 wherein the one or more additional quantitativeimmune-detection assays for assay of the quantity of the at least oneadditional marker are ELISA assays.
 36. The method of claim 35 whereinthe ELISA assays are sandwich ELISA assays.
 37. The method of claim 28further comprising the step of using age as a co-variate variable toincrease the sensitivity and specificity of the method.
 38. The methodof claim 28 wherein the quantitation of step (d) is digitally scannedand uploaded to the server of a medical provider.
 39. The method ofclaim 28 wherein the colorectal cancer is selected from the groupconsisting of stage I colorectal cancer, stage II colorectal cancer,stage IIIA colorectal cancer, stage IIIB colorectal cancer, stage IIICcolorectal cancer, and stage IV colorectal cancer.
 40. The method ofclaim 28 wherein the quantitative immune-detection assay is performedusing a magnetic bead.
 41. The method of claim 40 wherein thequantitative immune-detection assay is performed using detection ofphycoerythrin conjugated to streptavidin.
 42. A method for detectionand/or diagnosis of a precancerous condition of the digestive tract in asubject comprising: (a) obtaining a serum sample from the subject; (b)desialylating the serum sample; (c) contacting the desialylated serumsample with an antibody that binds haptoglobin to form a complex; and(d) quantitating the complex with a detectable lectin that bindsgalactose in order to detect or diagnose the precancerous condition ofthe digestive tract using a quantitative immune-detection assay, whereinthe presence of a galactose-containing haptoglobin glycoform isassociated with the presence of a precancerous condition of thedigestive tract, wherein the quantitative immune-detection assay is nota Western blot.
 43. The method of claim 42 wherein the precancerouscondition of the digestive tract is an adenomatous polyp.
 44. The methodof claim 43 wherein the adenomatous polyp is an advanced adenoma. 45.The method of claim 42, wherein the serum is diluted between about25-fold to about 50-fold prior to desialylation.
 46. The method of claim42, wherein the serum is diluted at least 50-fold prior todesialylation.
 47. The method of claim 42, wherein the desialylatedserum is further diluted to a final dilution of between about 100-foldand 150,000-fold; of between about 1,000-fold and about 150,000-fold, orabout 15,000-fold and about 150,000-fold before step (c).
 48. The methodof claim 47, wherein the desialylated serum is further diluted to afinal dilution of about 20,000-fold before step (c).
 49. The method ofclaim 42, wherein the quantitative immune-detection assay is an ELISAassay, a radioimmunoassay (RIA), an immunoradiometric assay, afluoroimmunoassay, a chemiluminescent immunoassay, a bioluminescentimmunoassay, an enzyme multiplied immunoassay (EMIT), a cloned enzymedonor immunoassay (CEDIA), an immuno-PCR assay, a phosphor immunoassay,a quantum dot immunoassay, a solid phase light-scattering immunoassay, asurface effect immunoassay, or an immunoassay employing lateral flowtest strips.
 50. The method of claim 49, wherein the quantitativeimmune-detection assay is an ELISA.
 51. The method of claim 50, whereinthe ELISA is a sandwich ELISA.
 52. The method of claim 42, whereindesialylating the serum comprises treating the serum with a mild acid.53. The method of claim 52, wherein the mild acid is H₂SO_(4.)
 54. Themethod of claim 42, wherein desialylating the serum comprises treatingthe serum with a neuraminidase.
 55. The method of claim 42, wherein theantibody that binds haptoglobin is a polyclonal antibody.
 56. The methodof claim 42, wherein the antibody that binds haptoglobin is bound to asubstrate or a magnetic bead.
 57. The method of claim 55, wherein theantibody that binds haptoglobin is a polyclonal antibody raised againstpurified human haptoglobin.
 58. The method of claim 42, wherein themethod further comprises a step of contacting the complex of step (c)with a wash solution prior to step (d).
 59. The method of claim 58,wherein the wash solution comprises a detergent.
 60. The method of claim42, wherein the detectable lectin that binds galactose is selected fromthe group consisting of Ricinus communis lectin, Sophora japonicalectin, Datura stramonium lectin, Erythrina cristagalli lectin, andLycopersicon esculentum lectin.
 61. The method of claim 60 wherein thedetectable lectin is Erythrina cristagalli lectin.
 62. The method ofclaim 42 wherein quantitating the complex with a detectable lectincomprises detecting an enzymatic activity.
 63. The method of claim 62,wherein the lectin is biotinylated or comprises a conjugated enzyme. 64.The method of claim 63 wherein the lectin is biotinylated and detectingthe complex comprises contacting the complex with an avidin-reporterconjugate.
 65. The method of claim 64 wherein the reporter is an enzyme.66. The method of claim 65 wherein the enzyme is selected from the groupconsisting of urease, alkaline phosphatase, horseradish peroxidase,glucose 6-phosphate dehydrogenase, β-galactosidase, and glucose oxidase.67. The method of claim 42 further comprising the step of using age as aco-variate variable to increase the sensitivity and specificity of themethod.
 68. The method of claim 42 wherein the quantitation of step (d)is digitally scanned and uploaded to the server of a medical provider.69. The method of claim 42 further comprising performance of apolypectomy to treat an adenomatous polyp.
 70. The method of claim 42wherein the quantitative immune-detection assay is performed using amagnetic bead.
 71. The method of claim 70 wherein the quantitativeimmune-detection assay is performed using detection of phycoerythrinconjugated to streptavidin.
 72. A method for detection and/or diagnosisof a precancerous condition of the digestive tract in a subjectcomprising: (a) obtaining a serum sample from the subject; (b)desialylating the serum sample; (c) contacting the desialylated serumsample with an antibody that binds haptoglobin to form a complex; (d)quantitating the complex with a detectable lectin that binds galactosein order to detect or diagnose a precancerous condition of the digestivetract using a quantitative immune-detection assay; and (e) assaying thesample for the quantity of at least one additional marker to confirm thedetection or diagnosis of a precancerous condition of the digestivetract using one or more additional quantitative immune-detection assays.73. The method of claim 72 wherein the precancerous condition of thedigestive tract is an adenomatous polyp.
 74. The method of claim 73wherein the adenomatous polyp is an advanced adenoma.
 75. The method ofclaim 72 wherein the at least one additional marker is at least one ofgalectin-3, a peptide derived from galectin-3, carcinoembryonic antigen(CEA), and CYFRA21-1.
 76. The method of claim 75 wherein the at leastone additional marker is galectin-3, CYFRA21-1, and CEA.
 77. The methodof claim 75 wherein the at least one additional marker is CYFRA21-1 andCEA.
 78. The method of claim 75 wherein the at least one additionalmarker is CYFRA21-1.
 79. The method of claim 75 wherein the at least oneadditional marker is CEA.
 80. The method of claim 75 wherein the one ormore additional quantitative immune-detection assays for assay of thequantity of the at least one additional marker are selected from thegroup consisting of an ELISA assay, a radioimmunoassay (RIA), animmunoradiometric assay, a fluoroimmunoassay, a chemiluminescentimmunoassay, a bioluminescent immunoassay, an enzyme multipliedimmunoassay (EMIT), a cloned enzyme donor immunoassay (CEDIA), animmuno-PCR assay, a phosphor immunoassay, a quantum dot immunoassay, asolid phase light-scattering immunoassay, a surface effect immunoassay,and an immunoassay employing lateral flow test strips.
 81. The method ofclaim 80 wherein the one or more additional quantitativeimmune-detection assays for assay of the quantity of the at least oneadditional marker are ELISA assays.
 82. The method of claim 81 whereinthe ELISA assays are sandwich ELISA assays.
 83. The method of claim 72further comprising the step of using age as a co-variate variable toincrease the sensitivity and specificity of the method.
 84. The methodof claim 72 wherein the quantitation of step (d) is digitally scannedand uploaded to the server of a medical provider.
 85. The method ofclaim 72 further comprising performance of a polypectomy to treat anadenomatous polyp.
 86. The method of claim 72 wherein the quantitativeimmune-detection assay for detection of the complex with a detectablelectin that binds galactose is an ELISA.
 87. The method of claim 72wherein the quantitative immune-detection assay for detection of thecomplex with a detectable lectin that binds galactose is performed usinga magnetic bead.
 88. The method of claim 87 wherein the quantitativeimmune detection assay for detection of the complex with a detectablelectin that binds galactose is performed using detection ofphycoerythrin conjugated to streptavidin.
 89. A method for detectingcolorectal cancer or a precancerous condition of the digestive tract ina subject comprising: (a) measuring the level of galectin-3 ligand andCYFRA21-1 in a serum sample from the subject; and (b) determiningwhether the subject has a colorectal cancer or a colorectalpre-cancerous lesion based on both the level of galectin-3 ligand andthe level of CYFRA21-1.
 90. A method for detecting colorectal cancer orcolorectal pre-cancerous lesions in a subject comprising: (a) measuringthe level of galectin-3 ligand in a serum sample from the subject; and(b) determining whether the subject has a colorectal cancer or acolorectal pre-cancerous lesion based on both the level of galectin-3ligand and the age of the subject.
 91. The method of claim 89 or 90,further comprising: (a) measuring the level of galectin-3 ligand andCYFRA21-1 in a blood sample from the subject; and (b) determiningwhether the subject has a colorectal cancer or a colorectalpre-cancerous lesion based on all of the level of galectin-3 ligand, thelevel of CYFRA21-1 and the age of the subject.
 92. The method of any oneof claims 89-91, wherein: (a) the method further comprises measuring thelevel of one additional marker in the blood sample selected from thegroup consisting of galectin-3, a peptide derived from galectin-3 andcarcinoembryonic antigen (CEA); and (b) the method further comprisesdetermining whether the subject has a colorectal cancer or a colorectalpre-cancerous lesion based on the level of said one additional marker.93. The method of any one of claims 89-91, wherein: (a) furthercomprises measuring the level of two additional markers in the bloodsample selected from the group consisting of galectin-3, a peptidederived from galectin-3 and carcinoembryonic antigen (CEA); and (b)further comprises determining whether the subject has a colorectalcancer or a colorectal pre-cancerous lesion based on the level of saidtwo additional markers.
 94. The method of claim 89 or 90, furthercomprising: (a) measuring the level of galectin-3 ligand, CYFRA21-1,galectin-3 and CEA in a blood sample from the subject; and (b)determining whether the subject has a colorectal cancer or a colorectalpre-cancerous lesion based on all of the level of galectin-3 ligand,CYFRA21-1, galectin-3, CEA and the age of the subject.
 95. The method ofany one of claims 89-94, wherein the blood sample is a serum sample. 96.The method of any one of claims 89-94, further comprising collecting theblood sample from the subject.
 97. The method of any one of claims89-94, wherein the method is a method for determining whether thesubject has a colorectal cancer.
 98. The method of claim 97, wherein thecolorectal cancer is selected from the group consisting of stage Icolorectal cancer, stage II colorectal cancer, stage IIIA colorectalcancer, stage IIIB colorectal cancer, stage IIIC colorectal cancer, andstage IV colorectal cancer.
 99. The method of any one of claims 89-94,wherein the method is a method for determining whether the subject has acolorectal pre-cancerous lesion.
 100. The method of claim 99, whereinthe colorectal pre-cancerous lesion is an adenomatous polyp.
 101. Themethod of claim 100, wherein the adenomatous polyp is an advancedadenoma.
 102. The method of any one of claims 89-101, wherein measuringa level of galectin-3 ligand comprises: (i) desialylating the bloodsample; (ii) contacting the desialylated sample with an antibody thatbinds haptoglobin to form a complex; and (iii) quantitating the complexwith a detectable lectin that binds galactose in order to measure thelevel of galectin-3 ligand in the sample.
 103. The method of claim 102,wherein the sample is diluted between about 25-fold to about 50-foldprior to desialylation.
 104. The method of claim 102, wherein the serumis diluted at least 50-fold prior to desialylation.
 105. The method ofclaim 102, wherein the desialylated serum is further diluted to a finaldilution of between about 100-fold and 150,000-fold; of between about1,000-fold and about 150,000-fold, or about 15,000-fold and about150,000-fold before step (ii).
 106. The method of claim 105, wherein thedesialylated serum is further diluted to a final dilution of about20,000-fold before step (ii).
 107. The method of claim 102, whereinquantitating the complex is by a quantitative immune-detection assay.108. The method of claim 107, wherein the quantitative immune-detectionassay is not a Western blot.
 109. The method of claim 107, wherein thequantitative immune-detection assay is an ELISA assay, aradioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay,a chemiluminescent immunoassay, a bioluminescent immunoassay, an enzymemultiplied immunoassay (EMIT), a cloned enzyme donor immunoassay(CEDIA), an immuno-PCR assay, a phosphor immunoassay, a quantum dotimmunoassay, a solid phase light-scattering immunoassay, a surfaceeffect immunoassay, or an immunoassay employing lateral flow teststrips.
 110. The method of claim 109, wherein the quantitativeimmune-detection assay is an ELISA.
 111. The method of claim 110,wherein the ELISA is a sandwich ELISA.
 112. The method of claim 102,wherein desialylating the sample comprises treating the serum with amild acid.
 113. The method of claim 112, wherein the mild acid is H₂SO₄.114. The method of claim 102, wherein desialylating the sample comprisestreating the serum with a neuraminidase.
 115. The method of claim 102,wherein the antibody that binds haptoglobin is a polyclonal antibody.116. The method of claim 102, wherein the antibody that bindshaptoglobin is bound to a substrate or a magnetic bead.
 117. The methodof claim 115, wherein the antibody that binds haptoglobin is apolyclonal antibody raised against purified human haptoglobin.
 118. Themethod of claim 102, wherein the method further comprises a step ofcontacting the complex of step (ii) with a wash solution prior to step(ii).
 119. The method of claim 118, wherein the wash solution comprisesa detergent.
 120. The method of claim 102, wherein the detectable lectinthat binds galactose is selected from the group consisting of Ricinuscommunis lectin, Sophora japonica lectin, Datura stramonium lectin,Erythrina cristagalli lectin, and Lycopersicon esculentum lectin. 121.The method of claim 120, wherein the detectable lectin is Erythrinacristagalli lectin.
 122. The method of claim 102, wherein quantitatingthe complex with a detectable lectin comprises detecting an enzymaticactivity.
 123. The method of claim 122, wherein the lectin isbiotinylated or comprises a conjugated enzyme.
 124. The method of claim123, wherein the lectin is biotinylated and detecting the complexcomprises contacting the complex with an avidin-reporter conjugate. 125.The method of any one of claims 89-94, wherein the measuring isautomated.
 126. The method of any one of claims 89-94, wherein measuringthe level comprises performing a quantitative immune-detection assay.127. The method of claim 126, wherein the quantitative immune-detectionassay is an ELISA assay, a radioimmunoassay (RIA), an immunoradiometricassay, a fluoroimmunoassay, a chemiluminescent immunoassay, abioluminescent immunoassay, an enzyme multiplied immunoassay (EMIT), acloned enzyme donor immunoassay (CEDIA), an immuno-PCR assay, a phosphorimmunoassay, a quantum dot immunoassay, a solid phase light-scatteringimmunoassay, a surface effect immunoassay or an immunoassay employinglateral flow test strips.
 128. The method of claim 127, wherein thequantitative immune-detection assays is an ELISA assay.
 129. The methodof claim 128, wherein the ELISA assay is a sandwich ELISA assay.
 130. Amethod of treating a subject comprising administering a colonoscopy to asubject determined to have a colorectal pre-cancerous lesion inaccordance with any one of claims 89-125.
 131. A method of treating asubject comprising performance of a polypectomy on a subject determinedto have a precancerous condition of the digestive tract in accordancewith any one of claims 89-125.
 132. The method of claim 131, wherein theprecancerous condition of the digestive tract is an adenomatous polyp.133. A method of treating a subject comprising administering ananti-cancer therapy to a subject determined to have a colorectal canceror a precancerous condition of the digestive tract in accordance withany one of claims 89-125.
 134. The method of claim 133, wherein thesubject has a precancerous condition and is treated with by anendoscopic treatment of adenomatous polyp.
 135. The method of claim 133,wherein the subject was determined to have a colorectal cancer.
 136. Themethod of claim 134, wherein the colorectal cancer is selected from thegroup consisting of stage I colorectal cancer, stage II colorectalcancer, stage IIIA colorectal cancer, stage IIIB colorectal cancer,stage IIIC colorectal cancer, and stage IV colorectal cancer.